CN101082623A - Preparation of detecting reagent of helicobacter pylori high virulence - Google Patents
Preparation of detecting reagent of helicobacter pylori high virulence Download PDFInfo
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- CN101082623A CN101082623A CN 200710009200 CN200710009200A CN101082623A CN 101082623 A CN101082623 A CN 101082623A CN 200710009200 CN200710009200 CN 200710009200 CN 200710009200 A CN200710009200 A CN 200710009200A CN 101082623 A CN101082623 A CN 101082623A
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Abstract
本发明公开了一种幽门螺杆菌高毒力株检测试剂的制备，属人类疾病临床诊断的试剂。即通过分子生物学手段及基因的克隆表达和表达产物的纯化，获得重组OipA抗原，利用此抗原检测临床血清标本中的相应抗体，同时分离培养胃活检标本的Hp，对Hp菌株进行oipA信号区基因的扩增和测序，比较抗体与基因检测的结果，从而评价重组抗原检测血清相应抗体的敏感性和特异性。获得幽门螺杆菌oipA毒力基因片段—oipA6的重组子：oipA6－pET－42a，将重组子转化BL－21细胞，获得高效表达的OipA6融合蛋白，分子量为：42KD，经鉴定具有良好的抗原性，用于检测病人血清OipA抗体的敏感性和特异性分别达到95.16％和95.83％。The invention discloses the preparation of a detection reagent for a highly virulent strain of Helicobacter pylori, belonging to a reagent for clinical diagnosis of human diseases. That is, the recombinant OipA antigen is obtained by means of molecular biology, gene cloning and expression, and the purification of the expression product. This antigen is used to detect the corresponding antibody in the clinical serum specimen, and at the same time, Hp is isolated and cultured in the gastric biopsy specimen, and the oipA signal region of the Hp strain is detected. Amplify and sequence the gene, compare the results of the antibody and the gene test, so as to evaluate the sensitivity and specificity of the recombinant antigen to detect the corresponding antibody in the serum. Obtained Helicobacter pylori oipA virulence gene fragment—oipA6 recombinant: oipA6-pET-42a, transformed the recombinant into BL-21 cells, obtained highly expressed OipA6 fusion protein, molecular weight: 42KD, and was identified as having good antigenicity , the sensitivity and specificity for detecting OipA antibody in patients' serum reached 95.16% and 95.83%, respectively.
Description
技术领域technical field
本发明涉及人类疾病临床诊断的试剂，具体的说是一种幽门螺杆菌高毒力株检测试剂的制备。The invention relates to a reagent for clinical diagnosis of human diseases, in particular to the preparation of a detection reagent for a highly virulent strain of Helicobacter pylori.
背景技术Background technique
人群中幽门螺杆菌(Helicobacter pylori，Hp)的感染率高，西方国家达50％，发展中国家可高达90％，但只有10％的感染者发展为消化性疾病。Hp感染的不同结局与Hp菌株间的毒力不同有关，毒力基因的异质性，决定Hp感染后是否导致炎症以及溃疡的发生。因而，应用重要的Hp毒力标志，进行高毒力菌株的鉴定，对于临床诊断及是否需要进行Hp根除性治疗具有极其重要的指导意义。The infection rate of Helicobacter pylori (Hp) in the population is high, reaching 50% in western countries and up to 90% in developing countries, but only 10% of infected people develop peptic diseases. The different outcomes of Hp infection are related to the difference in virulence among Hp strains, and the heterogeneity of virulence genes determines whether Hp infection leads to inflammation and ulceration. Therefore, using important Hp virulence markers to identify highly virulent strains has extremely important guiding significance for clinical diagnosis and the need for Hp eradication therapy.
目前对于Hp感染的诊断可以分为两种：即侵入性检查和非侵入性检查。侵入性检查主要是通过胃镜取胃粘膜标本作快速尿素酶诊断、细菌培养、PCR和组织学检查；非侵入性检查包括血清抗体的检测、尿素呼气试验、粪便抗原检测等。因侵入性检查造成的创伤性，尤其不能被儿童所接受，故而非侵入性检查更多受到青睐。但是这些非侵入性的检查方法，如尿素呼气试验、粪便抗原和血清UreB抗体检测仅是对Hp感染进行诊断，不能对高毒株进行鉴定，目前对高毒株Hp感染的鉴定主要是通过检测血清中抗Hp CagA抗体。Currently, the diagnosis of Hp infection can be divided into two types: invasive examination and non-invasive examination. Invasive examinations mainly include rapid urease diagnosis, bacterial culture, PCR, and histological examinations by taking gastric mucosa samples through endoscopy; non-invasive examinations include serum antibody detection, urea breath test, and stool antigen detection. Because of the trauma caused by invasive examinations, especially children cannot accept them, so non-invasive examinations are more favored. However, these non-invasive inspection methods, such as urea breath test, fecal antigen and serum UreB antibody detection, are only for the diagnosis of Hp infection, and cannot identify high-virulence strains. Currently, the identification of high-virulence strain Hp infection is mainly through Anti-Hp CagA antibody in serum was detected.
越来越多的流行病学调查发现，CagA阳性菌的感染率极高，在西方国家约占60％～80％，国内感染率更高，有些地区甚至达到90％以上，Lazebnik LB等的研究也提示cagA+vacAsl感染率高，很难确定其与疾病的关系。可见，以CagA来标志毒力株，已不能很好地满足临床的需要，加上目前的检测试剂盒敏感性和特异性不理想，阻止了临床上的广泛应用。More and more epidemiological surveys have found that the infection rate of CagA-positive bacteria is extremely high, accounting for about 60% to 80% in western countries, and the domestic infection rate is even higher, even reaching more than 90% in some areas. Lazebnik LB et al. It also suggested that the infection rate of cagA+vacAsl was high, and it was difficult to determine its relationship with the disease. It can be seen that the use of CagA to mark virulent strains can no longer meet the clinical needs, and the sensitivity and specificity of the current detection kits are not ideal, which prevents widespread clinical application.
发明内容Contents of the invention
为了克服现有的技术不足，本发明的目的是要提供一种幽门螺杆菌毒力基因oipA的重组抗原，为临床幽门螺杆菌高毒力株感染的诊断提供敏感、特异的检测试剂。In order to overcome the deficiencies of the existing technology, the purpose of the present invention is to provide a recombinant antigen of the virulence gene oipA of Helicobacter pylori, and provide sensitive and specific detection reagents for the diagnosis of clinical infection of highly virulent strains of Helicobacter pylori.
本发明解决其技术问题所采用的技术方案是：将通过分子生物学手段，通过基因的克隆表达及表达产物的纯化，获得重组OipA抗原，利用此抗原检测临床血清标本中的相应抗体，同时分离培养胃活检标本的Hp，对Hp菌株进行oipA信号区基因的扩增和测序，比较抗体与基因检测的结果，从而评价重组抗原检测血清相应抗体的敏感性和特异性的幽门螺杆菌高毒力株试剂的制备，其特征是：The technical scheme adopted by the present invention to solve its technical problems is: obtain the recombinant OipA antigen through molecular biological means, through the cloning expression of the gene and the purification of the expression product, use this antigen to detect the corresponding antibody in the clinical serum specimen, and separate Cultivate Hp from gastric biopsy specimens, amplify and sequence the oipA signal region gene of Hp strains, compare the results of antibody and gene detection, so as to evaluate the sensitivity and specificity of recombinant antigen detection serum corresponding antibodies High virulence of Helicobacter pylori The preparation of strain reagent is characterized in that:
一、材料和方法1. Materials and methods
1.菌株、质粒：幽门螺杆菌NCTC11637，大肠杆菌BL-21、载体pET-42a；1. Strains and plasmids: Helicobacter pylori NCTC11637, Escherichia coli BL-21, vector pET-42a;
2.引物：根据GeneBank的oipA的全长基因序列，经同源性分析，在开放读码框架之内设计带有酶切位点的六对引物，下游相同，上游不同，将oipA基因截断为依次减小的六个不同的片断；PCR产物及其相应引物分别为：oipA1：pF-1/pR；oipA2：pF-2/pR；oipA3：pF-3/pR；oipA4：pF-4/pR；oipA5：pF-5/pR；oipA6：pF-6/pR；PCR扩增产物大小分别为：oipA1：863bp；oipA2：803bp；oipA3：743bp；oipA4：683bp；oipA5：443bp；oipA6：204bp；引物，序列如下：2. Primers: According to the full-length gene sequence of oipA in GeneBank, six pairs of primers with enzyme cutting sites were designed within the open reading frame through homology analysis. The downstream is the same, but the upstream is different, and the oipA gene is truncated as Six different fragments that decrease in turn; PCR products and their corresponding primers are: oipA1: pF-1/pR; oipA2: pF-2/pR; oipA3: pF-3/pR; oipA4: pF-4/pR ; oipA5: pF-5/pR; oipA6: pF-6/pR; the sizes of PCR amplification products are: oipA1: 863bp; oipA2: 803bp; oipA3: 743bp; oipA4: 683bp; oipA5: 443bp; , the sequence is as follows:
pF-1：5′CGGGATCCGGATTTTATTTAGGTTTA 3′pF-1: 5′CGGGATCCGGATTTTATTTAGGTTTA 3′
pF-2：5′CGGGATCCGGCAAAAAAGCTTTAGCAG 3′pF-2: 5′CGGGATCCGGCAAAAAAGCTTTAGCAG 3′
pF-3：5′CGGGATCCTTATTCCCCGAACAAAACAC 3′pF-3: 5′
pF-4：5′CGGGATCCAAAGATTCAACAAAGATCGC 3′pF-4: 5′
pF-5：5′CGGGATCCCGAGCGTCCCAAGAATATGTTG 3′pF-5: 5′CGGGATCCCGAGCGTCCCAAGAATATGTTG 3′
pF-6：5′CGGGATCCGGCGTTCGTTGGAGCGGTG 3′pF-6: 5′CGGGATCCGGCGTTCGTTGGAGCGGTG 3′
pR：5′CGGAATTCATGTTTGTTTTTAAAGTTA 3′pR: 5′CGGAATTCATGTTTGTTTTTAAAGTTA 3′
另设计oipA信号区引物SF/SRAnother design of oipA signal region primer SF/SR
oipA SF5′TCTTAAAACCAAAGAAAAACC 3′oipA SF5′TCTTAAAAACCAAAGAAAAACC 3′
SR5′ACAGAACCAACGCCACCAA 3′SR5′ACAGAACCAACGCCACCAA 3′
3.幽门螺杆菌oipA基因片断原核表达重组子的构建3. Construction of prokaryotic expression recombinants of Helicobacter pylori oipA gene fragments
3.1oipA基因片断的获得：以幽门螺杆菌NCTC11637DNA为模板，通过PCR法获得oipA基因片断。按照胶回收试剂盒回收并纯化PCR产物，于-20℃保存备用。3.1 Obtaining the oipA gene fragment: the oipA gene fragment was obtained by PCR method using Helicobacter pylori NCTC11637 DNA as a template. The PCR product was recovered and purified according to the gel recovery kit, and stored at -20°C for future use.
3.2表达载体的获得：将含质粒pET-42a的菌株划线分离接种于LB选择培养基上，37℃培养10h。挑取单菌落接种于适量LB选择培养液中，振荡培养8-10h。用质粒抽提试剂盒，提取质粒pET-42a。3.2 Obtaining the expression vector: the strain containing the plasmid pET-42a was streaked and inoculated on LB selection medium, and cultured at 37°C for 10 h. Pick a single colony and inoculate it in an appropriate amount of LB selection medium, and shake it for 8-10 hours. Plasmid pET-42a was extracted using a plasmid extraction kit.
3.3oipA基因片断及载体的酶切和载体去磷酸化3.3 Enzyme digestion of oipA gene fragment and vector and vector dephosphorylation
以BamH I和EcoRl I双酶切oipA基因片断和pET-42a载体。按纯化试剂盒纯化酶切产物；纯化的载体去磷酸化，反应体系：The oipA gene fragment and the pET-42a vector were cut with BamH I and EcoRl I. Purify the digested product according to the purification kit; dephosphorylate the purified vector, and the reaction system:
CIP1μl、载体25μl、Buffer 35μl、ddH2O 19μl、total 50μl，反应条件：37℃水浴1h；65℃水浴20min终止反应，用纯化试剂盒对去磷酸化后的载体进行纯化。
3.4目的基因与表达载体的连接：各取3μl纯化后的目的片段和载体于1％的琼脂糖凝胶中100V电泳20min，根据电泳条带大致确定连接反应体系，载体和插入片段的摩尔比3∶1到1∶3。连接反应体系：3.4 Connection of the target gene and the expression vector: take 3 μl of the purified target fragment and the vector and electrophoresis in 1% agarose gel at 100V for 20 minutes, and roughly determine the connection reaction system and the molar ratio of the vector and the insert fragment according to the electrophoresis band 3 :1 to 1:3. Connection reaction system:
反应条件：4℃，16h；65℃水浴10min终止反应。Reaction conditions: 4°C, 16h; 65°C water bath for 10min to terminate the reaction.
连接产物分别为：pET-42a/oipA1；pET-42a/oipA2；pET-42a/oipA3；pET-42a/oipA4；pET-42a/oipA5；pET-42a/oipA6。The ligation products were: pET-42a/oipA1; pET-42a/oipA2; pET-42a/oipA3; pET-42a/oipA4; pET-42a/oipA5; pET-42a/oipA6.
3.5重组子的转化与筛选：将获得的6个重组质粒，分别转化入感受态细胞BL-21，挑取若干个转化单菌落，用开瑞质粒抽提试剂盒提取重组质粒。以重组质粒为模板，pET-42a为阴性对照的模板，HpNCTC11637为阳性对照的模板；以pF-1/pR；pF-2/pR；pF-3/pR；pF-4/pR；pF-5/pR；pF-6/pR为引物，PCR扩增鉴定重组质粒，同时重组质粒用BamH I和EcoR I双酶切鉴定，将PCR和酶切鉴定均为阳性的重组子送测序。3.5 Transformation and screening of recombinants: The obtained 6 recombinant plasmids were transformed into competent cells BL-21 respectively, several transformed single colonies were picked, and the recombinant plasmids were extracted with Carrier plasmid extraction kit. Use the recombinant plasmid as a template, pET-42a as a negative control template, and HpNCTC11637 as a positive control template; use pF-1/pR; pF-2/pR; pF-3/pR; pF-4/pR; pF-5 /pR; pF-6/pR were used as primers, and the recombinant plasmids were identified by PCR amplification. At the same time, the recombinant plasmids were identified by double digestion with BamH I and EcoR I, and the recombinants that were positive for both PCR and enzyme digestion were sent for sequencing.
4.幽门螺杆菌oipA基因片段的表达及产物纯化4. Expression of Helicobacter pylori oipA gene fragment and product purification
4.1重组子的表达、鉴定及表达条件优化4.1 Expression, identification and optimization of expression conditions of recombinants
用IPTG(终浓度为1.0mmol/L)诱导6个重组子的表达，诱导后，收集菌液，10000rpm，离心10min，弃去上清。细菌沉淀(1.0ml菌液量)加1XSDS上样缓冲液重悬混匀，沸水浴10min，10000g离心2min。取上清20μl进行SDS-PAGE电泳(分离胶浓度10％)，将凝胶上的蛋白条带电转移至硝酸纤维素(NC)膜，用抗GST Tag抗体，Western Breeze化学发光法试剂盒检测融合蛋白中的GST标签。The expression of the 6 recombinants was induced with IPTG (final concentration: 1.0 mmol/L). After induction, the bacterial liquid was collected, centrifuged at 10000 rpm for 10 min, and the supernatant was discarded. Bacterial pellet (1.0ml bacterial liquid volume) was resuspended in 1XSDS loading buffer and mixed evenly, in boiling water bath for 10min, and centrifuged at 10000g for 2min. Take 20 μl of the supernatant for SDS-PAGE electrophoresis (separation gel concentration 10%), transfer the protein bands on the gel to nitrocellulose (NC) membrane, and detect fusion with anti-GST Tag antibody and Western Breeze chemiluminescence assay kit GST tags in proteins.
分别以终浓度0.5、1、1.5、2mmol/L的IPTG诱导重组子表达4h，收集菌体进行SDS-PAGE电泳，图像分析系统分析表达量，确定最佳IPTG浓度。The recombinants were induced to express with final concentrations of 0.5, 1, 1.5, and 2 mmol/L IPTG for 4 hours, and the bacteria were collected for SDS-PAGE electrophoresis. The image analysis system analyzed the expression level to determine the optimal IPTG concentration.
分别用终浓度1.5mmol/L的IPTG诱导重组子的表达，诱导时间分别定为0.5、1、2、3、4、5、6h，收集菌体进行SDS-PAGE电泳，图像分析系统分析表达量，确定最佳诱导时间。IPTG with a final concentration of 1.5mmol/L was used to induce the expression of recombinants, and the induction time was set at 0.5, 1, 2, 3, 4, 5, and 6 hours, respectively, and the bacteria were collected for SDS-PAGE electrophoresis, and the image analysis system was used to analyze the expression level , to determine the optimal induction time.
4.2重组蛋白肽段的纯化4.2 Purification of recombinant protein peptides
用终浓度1.5mmol/L的IPTG诱导重组子表达4h，提取细菌包涵体，用8mol/L尿素裂解缓冲液彻底溶解包涵体。10000g离心20min，弃沉淀(含不被尿素溶解成分)，上清加入2XSDS上样缓冲液混匀，100℃水浴10min后进行SDS-PAGE电泳分析，继而用Ni-NTA His.Bind树脂亲和纯化，用Bradford方法测定纯化的重组蛋白的浓度。The expression of the recombinant was induced by IPTG with a final concentration of 1.5mmol/L for 4h, the bacterial inclusion bodies were extracted, and the inclusion bodies were completely dissolved with 8mol/L urea lysis buffer. Centrifuge at 10,000g for 20min, discard the precipitate (containing components not dissolved by urea), add 2XSDS loading buffer to the supernatant and mix well, and perform SDS-PAGE electrophoresis analysis after 10min in water bath at 100℃, and then use Ni-NTA His.Bind resin for affinity purification , The concentration of the purified recombinant protein was determined by the Bradford method.
4.3重组蛋白肽段的抗原性鉴定4.3 Antigenic identification of recombinant protein peptides
用Western-blot方法，以羊抗Hp多克隆抗体检测重组蛋白肽段的抗原性。取诱导表达的重组蛋白进行SDS-PAGE电泳，转移、丽春红染色；NC膜用5％脱脂牛奶4℃封闭过夜；以5％脱脂牛奶1∶400稀释羊抗Hp多克隆抗体，37℃，孵育2h；以5％脱脂牛奶1∶2000稀释AP标记的兔抗羊IgG，37℃，孵育1h；加入显色液NBT/BCIP孵育数min，条带出现后用双蒸水漂洗数次。The antigenicity of the recombinant protein peptides was detected by Western-blot method with goat anti-Hp polyclonal antibody. Take the induced recombinant protein for SDS-PAGE electrophoresis, transfer, and Ponceau staining; NC membrane was blocked overnight at 4°C with 5% skim milk; goat anti-Hp polyclonal antibody was diluted 1:400 with 5% skim milk, 37°C, Incubate for 2 hours; dilute AP-labeled rabbit anti-goat IgG with 5% skimmed milk 1:2000, incubate at 37°C for 1 hour; add chromogenic solution NBT/BCIP and incubate for several minutes, rinse with double distilled water several times after the band appears.
4.4筛选抗原性强的重组蛋白肽段4.4 Screening recombinant protein peptides with strong antigenicity
用间接ELISA法检测重组蛋白肽段抗原性的强弱。将纯化的重组蛋白肽段，Hp NCTC11637水煮蛋白(阳性对照)以及大肠杆菌BL-21水煮蛋白(阴性对照)以包被液调整至相同浓度，各取100μl包被酶标反应孔，同时以100μl包被液包被一个酶标反应孔做空白对照。置于4℃包被24h。弃去包被液，加封闭液37℃封闭2h，以洗涤液加满孔洗涤3次，每次3min，尽量弃尽孔内的液体。将羊抗Hp多克隆抗体用PBS做1∶500稀释，取100μl加入酶标反应孔，37℃孵育1h，洗涤3次，每次3min，将AP标记的兔抗羊IgG用PBS做1∶4000稀释，取100μl加入反应孔，37℃孵育40min，洗涤每孔加入100μl NBT/BCIP显色液，室温反应10-15min，采用450nm波长，测定OD值，以空白对照孔调零。Indirect ELISA was used to detect the antigenicity of recombinant protein peptides. Adjust the purified recombinant protein peptides, Hp NCTC11637 boiled protein (positive control) and Escherichia coli BL-21 boiled protein (negative control) to the same concentration with the coating solution, take 100 μl each to coat the enzyme-labeled reaction well, and at the same time Coat an enzyme-labeled reaction well with 100 μl of coating solution as a blank control. Placed at 4°C for coating for 24h. Discard the coating solution, add blocking solution at 37°C for 2 hours, fill up the well with washing solution and wash 3 times, each time for 3 minutes, and discard as much liquid as possible in the well. Dilute the goat anti-Hp polyclonal antibody 1:500 with PBS, take 100 μl into the enzyme-labeled reaction well, incubate at 37°C for 1 hour, wash 3 times, each time for 3 minutes, and make AP-labeled rabbit anti-goat IgG 1:4000 with PBS Dilute, add 100 μl to the reaction well, incubate at 37°C for 40 minutes, add 100 μl NBT/BCIP chromogenic solution to each well for washing, react at room temperature for 10-15 minutes, use 450nm wavelength to measure the OD value, and set to zero with the blank control well.
5.0ipA6重组抗原检测血清相应抗体及其评价5.0 ipA6 Recombinant Antigen Detection Serum Corresponding Antibody and Its Evaluation
5.1收集临床标本5.1 Collection of clinical specimens
收集活检胃组织标本和相应的血清标本及资料。活检标本取胃窦部粘膜组织，一份作快速尿素酶实验(临床常规检查)，一份置0.5ml布氏培养液中用于细菌分离培养(常规分离培养与鉴定)。同时抽取相应病人外周静脉血5ml，离心收集血清，分装后-20℃保存备用，用于检测血清抗体。Biopsy gastric tissue samples and corresponding serum samples and data were collected. The biopsy specimens were taken from gastric antrum mucosal tissue, one was used for rapid urease test (clinical routine examination), and the other was placed in 0.5ml Brooke's medium for bacterial isolation and culture (routine isolation, culture and identification). At the same time, 5ml of peripheral venous blood was drawn from the corresponding patient, the serum was collected by centrifugation, and stored at -20°C after aliquoting for use in the detection of serum antibodies.
5.2Hp临床分离菌株oipA和cagA基因PCR检测PCR detection of oipA and cagA genes of 5.2Hp clinical isolates
以上述分离获得的Hp(83株)DNA为模板，通过PCR法获得oipA基因信号区DNA。反应体系：Using the Hp (strain 83) DNA isolated above as a template, the signal region DNA of the oipA gene was obtained by PCR. reaction system:
10Xbuffer 5μl10Xbuffer 5μl
dNTP(10mmol/L each) 1μldNTP(10mmol/L each) 1μl
primer SF(20-40μmol/L) 0.5μlprimer SF(20-40μmol/L) 0.5μl
primer SR(20-40μmol/L) 0.5μlprimer SR(20-40μmol/L) 0.5μl
template(0.15μg) 5μltemplate(0.15μg) 5μl
DNA Polymerase(5u/μl) 0.5μlDNA Polymerase(5u/μl) 0.5μl
MgCL2(25mmol/L) 4μlMgCl 2 (25mmol/L) 4μl
ddH2O 33.5μlddH 2 O 33.5 μl
total 50μltotal 50μl
反应条件：94℃5min，(94℃30s，55℃1min，72℃1min)×30，72℃5minReaction conditions: 94°C for 5min, (94°C for 30s, 55°C for 1min, 72°C for 1min)×30, 72°C for 5min
5.3oipA信号区PCR产物测序分析5.3 Sequencing analysis of PCR products in oipA signal region
用PCR纯化试剂盒对oipA信号区PCR产物进行纯化， 纯化后的PCR产物送测定序列，测序结果用Clustalx和Bioedit软件进行分析。The PCR product of the oipA signal region was purified with a PCR purification kit, and the purified PCR product was sent for sequence determination, and the sequencing results were analyzed with Clustalx and Bioedit software.
5.4病人血清OipA抗体5.4 Patient serum OipA antibody
血清OipA抗体检测：以纯化的OipA6蛋白肽段(0.95mg/ml)，Hp11637水煮蛋白(阳性对照)、大肠杆菌BL-21水煮蛋白(阴性对照)以及包被液(空白对照)各100μl包被酶标反应孔；一抗孵育：将收集的血清标本用PBS做1∶100稀释，取100μl加入反应孔，37℃孵育1h；二抗孵育：将HRP标记的抗人IgG用PBS做1∶4000稀释，取100μl加入反应孔，37℃孵育40min；显色及结果判断：以TMB为显色液，每孔加入100μl反应10-15min，当阳性对照孔出现明显颜色变化后，立即加入50μl终止液终止反应，采用450nm波长，测定OD值，以空白对照孔调零。标本OD值大于等于2.1倍阴性对照OD值为阳性，标本OD值小于2.1倍阴性对照OD值为阴性。Serum OipA antibody detection: 100 μl each of purified OipA6 protein peptide (0.95 mg/ml), Hp11637 boiled protein (positive control), Escherichia coli BL-21 boiled protein (negative control) and coating solution (blank control) Coat enzyme-labeled reaction wells; primary antibody incubation: dilute the collected serum samples 1:100 with PBS, take 100 μl into the reaction wells, and incubate at 37°C for 1 hour; secondary antibody incubation: make HRP-labeled anti-human IgG in PBS for 1 hour : 4000 dilution, take 100 μl into the reaction well, incubate at 37°C for 40 minutes; color development and result judgment: use TMB as the color development solution, add 100 μl to each well and react for 10-15 minutes, when the positive control well shows obvious color change, immediately add 50 μl Terminate the reaction with the stop solution, measure the OD value with a wavelength of 450nm, and adjust to zero with the blank control well. The sample OD value is greater than or equal to 2.1 times the negative control OD value is positive, and the sample OD value is less than 2.1 times the negative control OD value is negative.
二、结果2. Results
1.oipA基因片段重组表达质粒的构建1. Construction of recombinant expression plasmid of oipA gene fragment
重组子(pET-42a/oipA1-6)的PCR和酶切鉴定结果，重组质粒PCR扩增产物，大小分别约为oipA1：860bp；oipA2：800bp；oipA3：740bp；oipA4：680bp；oipA5：440bp；oipA6：200bp。重组质粒BamH I和EcoRl I双酶切产物用1％琼脂糖凝胶电泳，可见均有2条带，大的条带均处于相同位置，大于3000bp；小的条带位置各不相同，依次减小，大小变化与相应的目的基因片断一致。The results of PCR and enzyme digestion identification of the recombinant (pET-42a/oipA1-6), the PCR amplification products of the recombinant plasmid, the size is about oipA1: 860bp; oipA2: 800bp; oipA3: 740bp; oipA4: 680bp; oipA5: 440bp; oipA6: 200bp. The recombinant plasmid BamH I and EcoRl I double-digested products were subjected to 1% agarose gel electrophoresis, and there were 2 bands, and the large bands were at the same position, greater than 3000 bp; the positions of the small bands were different, and the Small, the size change is consistent with the corresponding target gene fragment.
经PCR、酶切鉴定为阳性重组子，送测序，经DNAMAN软件分析，与相应的序列一致。It was identified as a positive recombinant by PCR and enzyme digestion, sent for sequencing, and analyzed by DNAMAN software, which was consistent with the corresponding sequence.
测得序列如下：The measured sequence is as follows:
oipA1：oipA1:
GGATTTTATTTAGGTTTAAATTTTCTAGAAGGAAGCTATATTCAAGGACAAGGTAGTATCGGCAAAAAGGATTTTATTTAGGTTTAAATTTTCTAGAAGGAAGCTATATTCAAGGACAAGGTAGTATCGGCAAAAA
AGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCCCGAACAAAAGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCCCGAACAAA
ACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGATCGCTAACACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGATCGCTAAC
CGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAGCTTGGGGTATAAATATTTTTTCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAGCTTGGGGTATAAATATTTTTT
GGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGGTTACCAACTTGGTGGCGTTGGTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTTCTCTTTTTTTCGGTTACCAACTTGGTGGCGTTGGTT
CTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCAATACGGATTTACTTATTAATTGGCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTTACCCTATGGTTTCAATACGGATTTACTTATTAATTGG
ACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAAACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAA
AGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAATAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAAT
CTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCTCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCT
TTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGATTCAATCAAGTCAAGAGCCACACAATTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGA
TGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTGGGGGTTATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTTCTAGTGTTGAATTGGGGGTTA
AAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTGAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTG
GTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA2：oipA2:
GGCAAAAAAGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCCGGCAAAAAAGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCC
CGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGACGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGA
TCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAGCTTGGGGTATAAATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTCAATGAACTCAGCTTGGGGTATAAA
TATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGGTTACCAACTTGGTGGTATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTTCTCTTTTTTTCGGTTACCAACTTGGTGG
CGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCAATACGGATTTACTTACGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTTACCCTATGGTTTCAATACGGATTTACTTA
TTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATA
TTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATTTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAAT
CAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATC
TCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGG
AGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATT
GGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATA
AAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA3：oipA3:
TTATTCCCCGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCTTATTCCCCGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTC
AACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAGCTTGGAACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTCAATGAACTCAGCTTGG
GGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGGTTACCAAGGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTTCTCTTTTTTTCGGTTACCAA
CTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCAATACGGACTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTACCCTATGGTTTCAATACGGA
TTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGC
TTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGTTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGG
CACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGCACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAG
TAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTCAGTTACAAGGGAAATTTGGCG
TTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGT
GTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATT
GGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA4：oipA4:
AAAGATTCAACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTAAAGATTCAACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACT
CAGCTTGGGGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGCAGCTTGGGGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCG
GTTACCAACTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCGTTACCAACTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTC
AATACGGATTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAATACGGATTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGT
AAAAGGGCTTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGAAAAGGGCTTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAAG
CATCAAGGCACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGCATCAAGGCACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGG
TTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAATTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAA
ATTTGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGATTTGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGG
GTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGG
GATAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACAGATAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACA
TTAATTAA
oipA5：oipA5:
CGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAAAGATATGACCGGCGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAAAGATATGACCGG
CAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAATCTTCAGGACTTGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAATCTTCAGGACTTG
TAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTCTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTC
AAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGATGAATACGATATAAGAGGCCACACAATTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGATGAATACGATAT
TGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGT
TTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTGGTGAGCGTTTATTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTGGTGAGCGTTTAT
CTCAACTATACATATAACTTTAAAAACAAACATTAACTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA6：oipA6:
GGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTC
TAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATATAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATA
AATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
2.幽门螺杆菌oipA基因片段的表达及产物纯化2. Expression of Helicobacter pylori oipA gene fragment and product purification
2.1蛋白表达：六个oipA基因片断表达后的预测大小分别为：32KD；30KD；27KD；25KD；16KD；7.5KD，含GST以及His的融合蛋白肽段预测大小分别约为67KD；65KD；62KD；60KD；51KD；42KD。经IPTG诱导后，SDS-PAGE电泳可见，各重组菌均有一条表达增加的条带，分子量与预期的融合蛋白一致；经图像分析系统测定，表达量分别占菌体总蛋白的30.10％；30.22％；30.01％；30.48％；30.58％；44.44％。表达的融合蛋白分别命名为OipA1；OipA2；OipA3；OipA4；OipA5；OipA6，空载体转化菌蛋白命名为OipAK。2.1 Protein expression: The predicted sizes of the six oipA gene fragments after expression are: 32KD; 30KD; 27KD; 25KD; 16KD; 7.5KD. The predicted sizes of the fusion protein peptides containing GST and His are about 67KD; 65KD; 62KD; 60KD; 51KD; 42KD. After being induced by IPTG, SDS-PAGE electrophoresis showed that each recombinant bacteria had a band with increased expression, and the molecular weight was consistent with the expected fusion protein; as determined by the image analysis system, the expression levels accounted for 30.10% of the total bacterial protein; 30.22 %; 30.01%; 30.48%; 30.58%; 44.44%. The expressed fusion proteins were named OipA1; OipA2; OipA3; OipA4; OipA5; OipA6, and the protein transformed with empty vector was named OipAK.
2.2表达产物鉴定2.2 Identification of expression products
重组蛋白肽段经SDS-PAGE电泳并转膜后，经过Western-Breeze化学发光的X光片上相应位置各有一条特异性条带，可见各蛋白肽段均可以被GST单克隆抗体识别，证明所表达的乃是预期的融合蛋白肽段。After the recombinant protein peptides were electrophoresed by SDS-PAGE and transferred to the membrane, there was a specific band at the corresponding position on the X-ray film after Western-Breeze chemiluminescence. It can be seen that each protein peptide can be recognized by the GST monoclonal antibody, proving that the What is expressed is the expected fusion protein peptide.
2.3表达条件的优化2.3 Optimization of expression conditions
终浓度0.5、1、1.5、2mmol/L的IPTG均能诱导重组蛋白的表达，SDS-PAGE电泳分析显示，当IPTG终浓度为1.5mmol/L时，表达量最大。分别诱导0.5、1、2、3、4、5、6h，重组蛋白均有表达。以OipA6为例，SDS-PAGE电泳后图像分析显示，表达量分别占菌体总蛋白41.08％；46.8％；49.3％；52.47％；54.33％；47.74％和46.87％。其中，以诱导4h，含量54.33％为最高。即当诱导时间为4h时，重组蛋白的表达量最大。IPTG with a final concentration of 0.5, 1, 1.5, and 2mmol/L could induce the expression of the recombinant protein. SDS-PAGE electrophoresis analysis showed that the expression was the largest when the final concentration of IPTG was 1.5mmol/L. After induction for 0.5, 1, 2, 3, 4, 5, and 6 hours, the recombinant protein was expressed. Taking OipA6 as an example, image analysis after SDS-PAGE electrophoresis showed that the expression levels accounted for 41.08%; 46.8%; 49.3%; 52.47%; 54.33%; 47.74% and 46.87% of the total bacterial protein respectively. Among them, the content of 54.33% was the highest after 4 hours of induction. That is, when the induction time is 4 hours, the expression of the recombinant protein is the largest.
2.4重组蛋白肽段的纯化2.4 Purification of recombinant protein peptides
包涵体的粗纯Crude purity of inclusion bodies
对重组蛋白包涵体进行初步纯化，SDS-PAGE电泳分析显示，当尿素浓度为8mol/L时，包涵体的溶解度最大，各蛋白肽段的纯度分别达到80％；82％；84％；83％；81％；90％。Preliminary purification of the recombinant protein inclusion body, SDS-PAGE electrophoresis analysis showed that when the urea concentration was 8mol/L, the solubility of the inclusion body was the largest, and the purity of each protein peptide reached 80%; 82%; 84%; 83% ; 81%; 90%.
亲和纯化(His-Tag)Affinity purification (His-Tag)
经初步纯化的包涵体进行亲和层析纯化，分别洗脱4次后，SDS-PAGE电泳分析显示，纯度分别可以达到：93％-96％，The preliminarily purified inclusion bodies were purified by affinity chromatography, and after elution for 4 times, SDS-PAGE electrophoresis analysis showed that the purity could reach: 93%-96%,
2.5重组蛋白肽段浓度测定2.5 Determination of recombinant protein peptide concentration
用Bradford方法测得的重组蛋白肽段的浓度见表1。The concentrations of recombinant protein peptides measured by the Bradford method are shown in Table 1.
表1：重组蛋白浓度Table 1: Recombinant Protein Concentrations
2.6重组蛋白肽段的抗原性2.6 Antigenicity of recombinant protein peptides
Western blot结果显示，NC膜上各有一条特异性条带，条带位置与重组蛋白肽段位置一致。说明重组蛋白肽段可以被羊抗Hp多克隆抗体识别，具有抗原性。The results of Western blot showed that there was a specific band on each NC membrane, and the position of the band was consistent with the position of the recombinant protein peptide. It shows that the recombinant protein peptide can be recognized by goat anti-Hp polyclonal antibody and has antigenicity.
2.7筛选抗原性强的重组蛋白肽段2.7 Screening of recombinant protein peptides with strong antigenicity
间接ELISA法检测重组蛋白肽段的抗原性，结果见表2。阳性对照Hp NCTC11637蛋白的吸光度最大，阴性对照大肠杆菌BL-21蛋白的吸光度最小，重组蛋白的吸光度从大到小依次为OipA6＞OipA5＞OipA4＞OipA3＞OipA2＞OipA1。表明同样条件下，OipA6的抗原性最强。The antigenicity of the recombinant protein peptide was detected by indirect ELISA, and the results are shown in Table 2. The positive control Hp NCTC11637 protein had the largest absorbance, the negative control Escherichia coli BL-21 protein had the smallest absorbance, and the absorbance of the recombinant protein in descending order was OipA6>OipA5>OipA4>OipA3>OipA2>OipA1. It shows that under the same conditions, OipA6 has the strongest antigenicity.
表2：重组蛋白ELISA的吸光度Table 2: Absorbance of recombinant protein ELISA
3.OipA6重组抗原检测血清相应抗体及其评价3. OipA6 recombinant antigen detection serum corresponding antibody and its evaluation
3.1临床标本分离oipA基因信号区序列分析3.1 Sequence analysis of signal region of oipA gene isolated from clinical specimens
临床标本分离培养获得83株Hp，PCR扩增其oipA信号区基因，66株细菌获得阳性结果，(随机显示6株细菌PCR结果)。将66株细菌的oipA基因PCR扩增产物进行序列分析，根据信号区AG(CT)重复数目的多少，判断oipA基因的开关状态：当信号区有6、9、(1+3)、(2+3)、(1+2)、(1+1+1)、(1+1+2)个CT双核苷酸重复时，基因为“开放”状态，可编码OipA蛋白，使机体产生相应的抗体；当信号区有5个或7个CT双核苷酸重复时，基因为“关闭”状态，不编码OipA蛋白。依此判断在66株细菌中，基因处于开放状态的有62株；基因处于关闭状态的0株；无法判断开关状态的有4株。相应信号区的CT(AG)重复数目、序列、基因的开关状态如表3。83 strains of Hp were isolated and cultured from clinical specimens, and the oipA signal region gene was amplified by PCR, and 66 strains of bacteria obtained positive results (PCR results of 6 strains of bacteria were randomly displayed). Sequence analysis was carried out on the oipA gene PCR amplification products of 66 strains of bacteria, and the switch state of the oipA gene was judged according to the number of repeats in the signal region AG (CT): when the signal region had 6, 9, (1+3), (2 When +3), (1+2), (1+1+1), (1+1+2) CT dinucleotide repeats, the gene is in the "open" state, which can encode OipA protein, so that the body can produce corresponding Antibody; when there are 5 or 7 CT dinucleotide repeats in the signal region, the gene is in the "off" state and does not encode OipA protein. According to this, among the 66 strains of bacteria, 62 strains had the gene in the open state; 0 strains had the gene in the closed state; and 4 strains could not determine the state of the switch. Table 3 shows the CT(AG) repeat number, sequence, and gene switch status of the corresponding signal region.
表3：66株Hp信号区AG(CT)重复序列分析结果Table 3: Analysis results of AG (CT) repeat sequence in the signal region of 66 strains of Hp
3.2抗原检测OipA抗体的敏感性和特异性3.2 Sensitivity and specificity of antigen detection OipA antibody
检测62例oipA信号区基因为开放状态(即可编码OipA，机体可产生抗体)的血清标本，及信号区PCR结果阴性和未感染Hp(Hp培养、血清CagA抗体、胃粘膜快速尿素酶检测同时阴性)的48例(即功能性oipA阴性)血清标本的OipA抗体，比较血清OipA抗体与oipA基因检测结果，如表4，结果显示，以OipA6作为抗原检测OipA抗体的敏感性为95.16％(59/62)，特异性为95.83％(46/48)，约登指数(Youden′s index)＝90.99％。Detect 62 cases of serum samples whose oipA signal region gene is in an open state (it can encode OipA, and the body can produce antibodies), and the PCR results of the signal region are negative and uninfected Hp (Hp culture, serum CagA antibody, gastric mucosa rapid urease detection at the same time Negative) of the OipA antibody of 48 cases (i.e. functional oipA negative) serum specimens, compare serum OipA antibody and oipA gene detection results, as shown in Table 4, the results show that the sensitivity of OipA antibody detection with OipA6 as antigen is 95.16% (59 /62), specificity was 95.83% (46/48), Youden's index (Youden's index) = 90.99%.
表4：OipA抗体检测与oipA基因检测结果比较Table 4: Comparison of OipA antibody detection and oipA gene detection results
本发明的有益效果是：获得幽门螺杆菌oipA毒力基因片段-oipA6的重组子：oipA6-pET-42a(oipA6序列如下)，将重组子转化BL-21细胞，获得高效表达的OipA6融合蛋白，分子量为：42KD，经鉴定具有良好的抗原性，用于检测病人血清OipA抗体的敏感性和特异性分别达到95.16％和95.83％。oipA6基因序列：The beneficial effects of the present invention are: obtaining the recombinant of Helicobacter pylori oipA virulence gene fragment-oipA6: oipA6-pET-42a (oipA6 sequence is as follows), transforming the recombinant into BL-21 cells, obtaining highly expressed OipA6 fusion protein, The molecular weight is 42KD, and it has been identified as having good antigenicity. The sensitivity and specificity for detecting the OipA antibody in patient serum reach 95.16% and 95.83%, respectively. oipA6 gene sequence:
GGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTC
TAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATATAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATA
AATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
附图说明Description of drawings
以下结合附图及实施例对本发明进行进一步的描述。The present invention will be further described below in conjunction with the accompanying drawings and embodiments.
图1是重组质粒PCR鉴定结果。Figure 1 is the result of PCR identification of the recombinant plasmid.
图中M：DNA Marker DL3000；M in the picture: DNA Marker DL3000;
1-6：pET-42a/oipA1-6；K：pET-42a。1-6: pET-42a/oipA1-6; K: pET-42a.
图2是重组质粒酶切产物电泳。Figure 2 is the electrophoresis of the digested product of the recombinant plasmid.
图中M：DNA Marker DL3000；1-6：pET-42a/oipA1-6。In the figure, M: DNA Marker DL3000; 1-6: pET-42a/oipA1-6.
图3是OipA1-6融合蛋白肽段的SDS-PAGE电泳图。Figure 3 is the SDS-PAGE electrophoresis of the OipA1-6 fusion protein peptide.
图中1～6：OipA1～6，respectively；K：OipAK；M：Protein Marker。Figures 1-6: OipA1-6, respectively; K: OipAK; M: Protein Marker.
图4是重组融合蛋白肽段的鉴定。Figure 4 is the identification of recombinant fusion protein peptides.
图中1：OipA1；2：OipA2；3：OipA3；4：OipA4；5：OipA5；6：OipA6。In the figure 1: OipA1; 2: OipA2; 3: OipA3; 4: OipA4; 5: OipA5; 6: OipA6.
图5是不同时间诱导OipA6的表达。Figure 5 is the expression of OipA6 induced at different times.
图中0：induced for Oh；0.5：induced for 0.5h；In the figure 0: induced for Oh; 0.5: induced for 0.5h;
1～6：induced for 1～6h respectively。1～6: induced for 1～6h respectively.
图6是重组蛋白肽段包涵体初步纯化。Figure 6 is the preliminary purification of recombinant protein peptide inclusion body.
图中K：OipAK after induction；K’：inclusion body of OipAK；1～6：OipA1～6 afterinduction；1’～6’：inclusion body of OipA1-6；M：Marker。In the figure K: OipAK after induction; K': inclusion body of OipAK; 1~6: OipA1~6 after induction; 1'~6': inclusion body of OipA1-6; M: Marker.
图7是重组蛋白肽段亲和纯化。Figure 7 is the affinity purification of recombinant protein peptides.
图中1，5，6：OipA1、5、6 after induction；1, 5, 6 in the figure: OipA1, 5, 6 after induction;
1’，5’，6’：OipA1、5、6 after affinity purification1', 5', 6': OipA1, 5, 6 after affinity purification
图8是重组蛋白肽段抗原性检测。Figure 8 is the detection of antigenicity of recombinant protein peptides.
图中1：OipA1；2：OipA2；3：OipA3；In the figure 1: OipA1; 2: OipA2; 3: OipA3;
4：OipA4；5：OipA5；6：OipA6。4: OipA4; 5: OipA5; 6: OipA6.
图9是oipA基因信号区PCR扩增产物。Figure 9 is the PCR amplification product of the oipA gene signal region.
图中M：Marker；1～6：clinical sample。In the figure, M: Marker; 1-6: clinical sample.
具体实施方式Detailed ways
实施例1：Example 1:
一、材料和方法1. Materials and methods
1.菌株、质粒：幽门螺杆菌NCTC11637购自中国疾病预防控制中心传染病研究所，大肠杆菌BL-21、载体pET-42a购自Invitrogen公司1. Strains and plasmids: Helicobacter pylori NCTC11637 was purchased from the Institute of Infectious Diseases of the Chinese Center for Disease Control and Prevention, Escherichia coli BL-21 and vector pET-42a were purchased from Invitrogen
2.引物：由上海博亚生物工程有限公司合成。根据GeneBank公布的oipA的全长基因序列，经同源性分析，在开放读码框架之内设计带有酶切位点的六对引物，下游相同，上游不同，将oipA基因截断为依次减小的六个不同的片断。PCR产物及其相应引物分别为：oipA1：pF-1/pR；oipA2：pF-2/pR；oipA3：pF-3/pR；oipA4：pF-4/pR；oipA5：pF-5/pR；oipA6：pF-6/pR。PCR扩增产物大小分别为：oipA1：863bp；oipA2：803bp；oipA3：743bp；oipA4：683bp；oipA5：443bp；oipA6：204bp。引物，序列如下：2. Primers: synthesized by Shanghai Boya Bioengineering Co., Ltd. According to the full-length gene sequence of oipA published by GeneBank, after homology analysis, six pairs of primers with enzyme cutting sites were designed within the open reading frame. of six different pieces. The PCR products and their corresponding primers are: oipA1: pF-1/pR; oipA2: pF-2/pR; oipA3: pF-3/pR; oipA4: pF-4/pR; oipA5: pF-5/pR; oipA6 : pF-6/pR. The sizes of PCR amplification products were: oipA1: 863bp; oipA2: 803bp; oipA3: 743bp; oipA4: 683bp; oipA5: 443bp; oipA6: 204bp. Primers, the sequence is as follows:
pF-1：5′CGGGATCCGGATTTTATTTAGGTTTA 3′pF-1: 5′
pF-2：5′CGGGATCCGGCAAAAAAGCTTTAGCAG 3′pF-2: 5′
pF-3：5′CGGGATCCTTATTCCCCGAACAAAACAC 3′pF-3: 5′
pF-4：5′CGGGATCCAAAGATTCAACAAAGATCGC 3′pF-4: 5′
pF-5：5′CGGGATCCCGAGCGTCCCAAGAATATGTTG 3′pF-5: 5′
pF-6：5′CGGGATCCGGCGTTCGTTGGAGCGGTG 3′pF-6: 5′
pR：5′CGGAATTCATGTTTGTTTTTAAAGTTA 3′pR: 5′
另设计oipA信号区引物SF/SRAnother design of oipA signal region primer SF/SR
oipA SF5′TCTTAAAACCAAAGAAAAACC 3′oipA
SR 5′ACAGAACCAACGCCACCAA 3′
3.幽门螺杆菌oipA基因片断原核表达重组子的构建3. Construction of prokaryotic expression recombinants of Helicobacter pylori oipA gene fragments
3.1 oipA基因片断的获得：以幽门螺杆菌NOTC11637 DNA为模板，通过PCR法获得oipA基因片断。按照上海中科开瑞工程有限公司胶回收试剂盒回收并纯化PCR产物，于-20℃保存备用。3.1 Obtaining the oipA gene fragment: using Helicobacter pylori NOTC11637 DNA as a template, the oipA gene fragment was obtained by PCR. The PCR products were recovered and purified according to the glue recovery kit of Shanghai Zhongke Kairui Engineering Co., Ltd., and stored at -20°C for later use.
3.2表达载体的获得：将含质粒pET-42a的菌株划线分离接种于LB选择培养基上，37℃培养10h。挑取单菌落接种于适量LB选择培养液中，振荡培养8-10h。用上海中科开瑞工程有限公司质粒抽提试剂盒，提取质粒pET-42a。3.2 Obtaining the expression vector: the strain containing the plasmid pET-42a was streaked and inoculated on LB selection medium, and cultured at 37°C for 10 h. Pick a single colony and inoculate it in an appropriate amount of LB selection medium, and shake it for 8-10 hours. Plasmid pET-42a was extracted with the plasmid extraction kit of Shanghai Zhongke Kairui Engineering Co., Ltd.
3.3 oipA基因片断及载体的酶切和载体去磷酸化3.3 Digestion of oipA gene fragment and vector and vector dephosphorylation
以BamH I和EcoRl I双酶切oipA基因片断和pET-42a载体。按照上海中科开瑞工程有限公司纯化试剂盒纯化酶切产物。纯化的载体去磷酸化，反应体系：CIP1μl、载体25μl、Buffer35μl、ddH2O19μl、total50μl，反应条件：37℃水浴1h；65℃水浴20min终止反应，用纯化试剂盒对去磷酸化后的载体进行纯化。The oipA gene fragment and the pET-42a vector were digested with BamH I and EcoRl I. The digested products were purified according to the purification kit of Shanghai Zhongke Kairui Engineering Co., Ltd. Dephosphorylation of the purified carrier, reaction system: CIP1μl, carrier 25μl, Buffer35μl, ddH 2 O19μl, total50μl, reaction conditions: 37°C water bath for 1h; 65°C water bath for 20min to stop the reaction, use the purification kit to carry out the dephosphorylated carrier purification.
3.4目的基因与表达载体的连接：各取3μl纯化后的目的片段和载体于1％的琼脂糖凝胶中100V电泳20min，根据电泳条带大致确定连接反应体系，载体和插入片段的摩尔比3∶1到1∶3。连接反应体系：3.4 Connection of the target gene and the expression vector: take 3 μl of the purified target fragment and the vector and electrophoresis in 1% agarose gel at 100V for 20 minutes, and roughly determine the connection reaction system and the molar ratio of the vector and the insert fragment according to the electrophoresis band 3 :1 to 1:3. Connection reaction system:
反应条件：4℃，16h；65℃水浴10min终止反应。Reaction conditions: 4°C, 16h; 65°C water bath for 10min to terminate the reaction.
连接产物分别为：pET-42a/oipA1；pET-42a/oipA2；pET-42a/oipA3；pET-42a/oipA4；pET-42a/oipA5；pET-42a/oipA6。The ligation products were: pET-42a/oipA1; pET-42a/oipA2; pET-42a/oipA3; pET-42a/oipA4; pET-42a/oipA5; pET-42a/oipA6.
3.5重组子的转化与筛选：将上述获得的6个重组质粒，分别转化入感受态细胞BL-21，挑取若干个转化单菌落，用上海中科开瑞质粒抽提试剂盒提取重组质粒。以重组质粒为模板，pET-42a为阴性对照的模板，HpNCTC11637为阳性对照的模板；以pF-1/pR；pF-2/pR；pF-3/pR；pF-4/pR；pF-5/pR；pF-6/pR为引物，PCR扩增鉴定重组质粒，同时重组质粒用BamH I和EcoR I双酶切鉴定，将PCR和酶切鉴定均为阳性的重组子送上海博亚生物技术有限公司测序。3.5 Transformation and screening of recombinants: Transform the 6 recombinant plasmids obtained above into competent cells BL-21, pick several transformed single colonies, and extract the recombinant plasmids with Shanghai Zhongke Carrier Plasmid Extraction Kit. Use the recombinant plasmid as a template, pET-42a as a negative control template, and HpNCTC11637 as a positive control template; use pF-1/pR; pF-2/pR; pF-3/pR; pF-4/pR; pF-5 /pR; pF-6/pR as primers, PCR amplification and identification of recombinant plasmids, and at the same time recombinant plasmids were identified by double enzyme digestion with BamH I and EcoR I, and recombinants that were positive in both PCR and enzyme digestion identification were sent to Shanghai Boya Biotechnology Ltd. Sequencing.
4.幽门螺杆菌oipA基因片段的表达及产物纯化4. Expression of Helicobacter pylori oipA gene fragment and product purification
4.1重组子的表达、鉴定及表达条件优化4.1 Expression, identification and optimization of expression conditions of recombinants
用IPTG(终浓度为1.0mmol/L)诱导上述6个重组子的表达，诱导后，收集菌液，10000rpm，离心10min，弃去上清。细菌沉淀(1.0ml菌液量)加1XSDS上样缓冲液重悬混匀，沸水浴10min，10000g离心2min。取上清20μl进行SDS-PAGE电泳(分离胶浓度10％)，将凝胶上的蛋白条带电转移至硝酸纤维素(NC)膜，用抗GST Tag抗体，Western Breeze化学发光法试剂盒检测融合蛋白中的GST标签。The expression of the above six recombinants was induced with IPTG (final concentration: 1.0 mmol/L). After induction, the bacterial liquid was collected, centrifuged at 10000 rpm for 10 min, and the supernatant was discarded. Bacterial pellet (1.0ml bacterial liquid volume) was resuspended in 1XSDS loading buffer and mixed evenly, in boiling water bath for 10min, and centrifuged at 10000g for 2min. Take 20 μl of the supernatant for SDS-PAGE electrophoresis (separation gel concentration 10%), transfer the protein bands on the gel to nitrocellulose (NC) membrane, and detect fusion with anti-GST Tag antibody and Western Breeze chemiluminescence assay kit GST tags in proteins.
分别以终浓度0.5、1、1.5、2mmol/L的IPTG诱导重组子表达4h，收集菌体进行SDS-PAGE电泳，图像分析系统分析表达量，确定最佳IPTG浓度。The recombinants were induced to express with final concentrations of 0.5, 1, 1.5, and 2 mmol/L IPTG for 4 hours, and the bacteria were collected for SDS-PAGE electrophoresis. The image analysis system analyzed the expression level to determine the optimal IPTG concentration.
分别用终浓度1.5mmol/L的IPTG诱导重组子的表达，诱导时间分别定为0.5、1、2、3、4、5、6h，收集菌体进行SDS-PAGE电泳，图像分析系统分析表达量，确定最佳诱导时间。IPTG with a final concentration of 1.5mmol/L was used to induce the expression of recombinants, and the induction time was set at 0.5, 1, 2, 3, 4, 5, and 6 hours, respectively, and the bacteria were collected for SDS-PAGE electrophoresis, and the image analysis system was used to analyze the expression level , to determine the optimal induction time.
4.2重组蛋白肽段的纯化4.2 Purification of recombinant protein peptides
用终浓度1.5mmol/L的IPTG诱导重组子表达4h，提取细菌包涵体，用8mol/L尿素裂解缓冲液彻底溶解包涵体。10000g离心20min，弃沉淀(含不被尿素溶解成分)，上清加入2XSDS上样缓冲液混匀，100℃水浴10min后进行SDS-PAGE电泳分析，继而用Ni-NTA His.Bind树脂亲和纯化，用Bradford方法测定纯化的重组蛋白的浓度。The expression of the recombinant was induced by IPTG with a final concentration of 1.5mmol/L for 4h, the bacterial inclusion bodies were extracted, and the inclusion bodies were completely dissolved with 8mol/L urea lysis buffer. Centrifuge at 10,000g for 20min, discard the precipitate (containing components not dissolved by urea), add 2XSDS loading buffer to the supernatant and mix well, and perform SDS-PAGE electrophoresis analysis after 10min in water bath at 100℃, and then use Ni-NTA His.Bind resin for affinity purification , The concentration of the purified recombinant protein was determined by the Bradford method.
4.3重组蛋白肽段的抗原性鉴定4.3 Antigenic identification of recombinant protein peptides
用Western-blot方法，以羊抗Hp多克隆抗体检测重组蛋白肽段的抗原性。取诱导表达的重组蛋白进行SDS-PAGE电泳，转移、丽春红染色；NC膜用5％脱脂牛奶4℃封闭过夜；以5％脱脂牛奶1∶400稀释羊抗Hp多克隆抗体，37℃，孵育2h；以5％脱脂牛奶1∶2000稀释AP标记的兔抗羊IgG，37℃，孵育1h；加入显色液NBT/BCIP孵育数min，条带出现后用双蒸水漂洗数次。The antigenicity of the recombinant protein peptides was detected by Western-blot method with goat anti-Hp polyclonal antibody. Take the induced recombinant protein for SDS-PAGE electrophoresis, transfer, and Ponceau staining; NC membrane was blocked overnight at 4°C with 5% skim milk; goat anti-Hp polyclonal antibody was diluted 1:400 with 5% skim milk, 37°C, Incubate for 2 hours; dilute AP-labeled rabbit anti-goat IgG with 5% skimmed milk 1:2000, incubate at 37°C for 1 hour; add chromogenic solution NBT/BCIP and incubate for several minutes, rinse with double distilled water several times after the band appears.
4.4筛选抗原性强的重组蛋白肽段4.4 Screening recombinant protein peptides with strong antigenicity
用间接ELISA法检测重组蛋白肽段抗原性的强弱。将纯化的重组蛋白肽段，Hp NCTC11637水煮蛋白(阳性对照)以及大肠杆菌BL-21水煮蛋白(阴性对照)以包被液调整至相同浓度，各取100μl包被酶标反应孔，同时以100μl包被液包被一个酶标反应孔做空白对照。置于4℃包被24h。弃去包被液，加封闭液37℃封闭2h，以洗涤液加满孔洗涤3次，每次3min，尽量弃尽孔内的液体。将羊抗Hp多克隆抗体用PBS做1∶500稀释，取100μl加入酶标反应孔，37℃孵育1h，洗涤3次，每次3min，将AP标记的兔抗羊IgG用PBS做1∶4000稀释，取100μl加入反应孔，37℃孵育40min，洗涤每孔加入100μl NBT/BCIP显色液，室温反应10-15min，采用450nm波长，测定OD值，以空白对照孔调零。Indirect ELISA was used to detect the antigenicity of recombinant protein peptides. Adjust the purified recombinant protein peptides, Hp NCTC11637 boiled protein (positive control) and Escherichia coli BL-21 boiled protein (negative control) to the same concentration with the coating solution, take 100 μl each to coat the enzyme-labeled reaction well, and at the same time Coat an enzyme-labeled reaction well with 100 μl of coating solution as a blank control. Placed at 4°C for coating for 24h. Discard the coating solution, add blocking solution at 37°C for 2 hours, fill up the well with washing solution and wash 3 times, each time for 3 minutes, and discard as much liquid as possible in the well. Dilute the goat anti-Hp polyclonal antibody 1:500 with PBS, take 100 μl into the enzyme-labeled reaction well, incubate at 37°C for 1 hour, wash 3 times, each time for 3 minutes, and make AP-labeled rabbit anti-goat IgG 1:4000 with PBS Dilute, add 100 μl to the reaction well, incubate at 37°C for 40 minutes, add 100 μl NBT/BCIP chromogenic solution to each well for washing, react at room temperature for 10-15 minutes, use 450nm wavelength to measure the OD value, and set to zero with the blank control well.
5.OipA6重组抗原检测血清相应抗体及其评价5. OipA6 recombinant antigen detection serum corresponding antibody and its evaluation
5.1收集临床标本5.1 Collection of clinical specimens
收集285份活检胃组织标本和相应的血清标本，同时收集患者的临床资料(性别、年龄、临床诊断、既往消化道疾病史)和病理诊断结果。285份标本中男157例、女128例，平均年龄44.4岁。活检标本取胃窦部粘膜组织，一份作快速尿素酶实验(临床常规检查)，一份置0.5ml布氏培养液中用于细菌分离培养(常规分离培养与鉴定)。同时抽取相应病人外周静脉血5ml，离心收集血清，分装后-20℃保存备用，用于检测血清抗体。A total of 285 biopsied gastric tissue specimens and corresponding serum specimens were collected, and the clinical data (sex, age, clinical diagnosis, previous history of gastrointestinal diseases) and pathological diagnosis results of the patients were collected. Among the 285 samples, there were 157 males and 128 females, with an average age of 44.4 years. The biopsy specimens were taken from gastric antrum mucosal tissue, one was used for rapid urease test (clinical routine examination), and the other was placed in 0.5ml Brooke's medium for bacterial isolation and culture (routine isolation, culture and identification). At the same time, 5ml of peripheral venous blood was drawn from the corresponding patient, the serum was collected by centrifugation, and stored at -20°C after aliquoting for use in the detection of serum antibodies.
5.2Hp临床分离菌株oipA和cagA基因PCR检测PCR detection of oipA and cagA genes of 5.2Hp clinical isolates
以上述分离获得的Hp(83株)DNA为模板，通过PCR法获得oipA基因信号区DNA。反应体系：Using the Hp (strain 83) DNA isolated above as a template, the signal region DNA of the oipA gene was obtained by PCR. reaction system:
10Xbuffer 5μl10Xbuffer 5μl
dNTP(10mmol/L each) 1μldNTP(10mmol/L each) 1μl
primer SF(20-40μmol/L) 0.5μlprimer SF (20-40μmol/L) 0.5μl
primer SR(20-40μmol/L) 0.5μlprimer SR(20-40μmol/L) 0.5μl
template(0.15μg) 5μltemplate(0.15μg) 5μl
DNA Polymerase(5u/μl) 0.5μlDNA Polymerase(5u/μl) 0.5μl
MgCL2(25mmol/L) 4μlMgCl 2 (25mmol/L) 4μl
ddH2O 33.5μlddH 2 O 33.5 μl
total 50μltotal 50μl
反应条件：94℃5min，(94℃30s，55℃1min，72℃1min)×30，72℃5minReaction conditions: 94°C for 5min, (94°C for 30s, 55°C for 1min, 72°C for 1min)×30, 72°C for 5min
5.3 oipA信号区PCR产物测序分析5.3 PCR product sequencing analysis of oipA signal region
用上海中科开瑞工程有限公司PCR纯化试剂盒对oipA信号区PCR产物进行纯化，纯化后的PCR产物送上海博亚生物技术有限公司测定序列，测序结果用Clustalx和Bioedit软件进行分析。The PCR product of the oipA signal region was purified with a PCR purification kit from Shanghai Zhongke Kairui Engineering Co., Ltd. The purified PCR product was sent to Shanghai Boya Biotechnology Co., Ltd. for sequencing, and the sequencing results were analyzed with Clustalx and Bioedit software.
5.4病人血清OipA抗体5.4 Patient serum OipA antibody
血清OipA抗体检测：以纯化的OipA6蛋白肽段(0.95mg/ml)，Hp11637水煮蛋白(阳性对照)、大肠杆菌BL-21水煮蛋白(阴性对照)以及包被液(空白对照)各100μl包被酶标反应孔；一抗孵育：将收集的血清标本用PBS做1∶100稀释，取100μl加入反应孔，37℃孵育1h；二抗孵育：将HRP标记的抗人IgG用PBS做1∶4000稀释，取100μl加入反应孔，37℃孵育40min；显色及结果判断：以TMB为显色液，每孔加入100μl反应10-15min，当阳性对照孔出现明显颜色变化后，立即加入50μl终止液终止反应，采用450nm波长，测定OD值，以空白对照孔调零。标本OD值大于等于2.1倍阴性对照OD值为阳性，标本OD值小于2.1倍阴性对照OD值为阴性。Serum OipA antibody detection: 100 μl each of purified OipA6 protein peptide (0.95 mg/ml), Hp11637 boiled protein (positive control), Escherichia coli BL-21 boiled protein (negative control) and coating solution (blank control) Coat enzyme-labeled reaction wells; primary antibody incubation: dilute the collected serum samples 1:100 with PBS, take 100 μl into the reaction wells, and incubate at 37°C for 1 hour; secondary antibody incubation: make HRP-labeled anti-human IgG in PBS for 1 hour : 4000 dilution, take 100 μl into the reaction well, incubate at 37°C for 40 minutes; color development and result judgment: use TMB as the color development solution, add 100 μl to each well and react for 10-15 minutes, when the positive control well shows obvious color change, immediately add 50 μl Terminate the reaction with the stop solution, measure the OD value with a wavelength of 450nm, and adjust to zero with the blank control well. The sample OD value is greater than or equal to 2.1 times the negative control OD value is positive, and the sample OD value is less than 2.1 times the negative control OD value is negative.
二、结果2. Results
1.oipA基因片段重组表达质粒的构建1. Construction of recombinant expression plasmid of oipA gene fragment
图1、2分别为重组子(pET-42a/oipA1-6)的PCR和酶切鉴定结果，重组质粒PCR扩增产物，大小分别约为oipA1：860bp；oipA2：800bp；oipA3：740bp；oipA4：680bp；oipA5：440bp；oipA6：200bp，与之前所得的目的基因片断大小基本一致。重组质粒BamH I和EcoRl I双酶切产物用1％琼脂糖凝胶电泳，可见均有2条带，大的条带均处于相同位置，大于3000bp；小的条带位置各不相同，依次减小，大小变化与相应的目的基因片断一致。Figures 1 and 2 are the PCR and enzyme digestion identification results of the recombinant (pET-42a/oipA1-6), respectively, and the PCR amplification products of the recombinant plasmid, the size of which is about oipA1: 860bp; oipA2: 800bp; oipA3: 740bp; oipA4: 680bp; oipA5: 440bp; oipA6: 200bp, which are basically the same size as the target gene fragment obtained before. The recombinant plasmid BamH I and EcoRl I double-digested products were subjected to 1% agarose gel electrophoresis, and there were 2 bands, and the large bands were at the same position, greater than 3000 bp; the positions of the small bands were different, and the Small, the size change is consistent with the corresponding target gene fragment.
经PCR、酶切鉴定为阳性重组子，送测序，经DNAMAN软件分析，与相应的序列一致。It was identified as a positive recombinant by PCR and enzyme digestion, sent for sequencing, and analyzed by DNAMAN software, which was consistent with the corresponding sequence.
测得序列如下：The measured sequence is as follows:
oipA1：oipA1:
GGATTTTATTTAGGTTTAAATTTTCTAGAAGGAAGCTATATTCAAGGACAAGGTAGTATCGGCAAAAAGGATTTTATTTAGGTTTAAATTTTCTAGAAGGAAGCTATATTCAAGGACAAGGTAGTATCGGCAAAAA
AGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCCCGAACAAAAGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCCCGAACAAA
ACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGATCGCTAACACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGATCGCTAAC
CGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAG℃TTGGGGTATAAATATTTTTTCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAG℃TTGGGGTATAAATATTTTTT
GGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGGTTACCAACTTGGTGGCGTTGGTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTTCTCTTTTTTTCGGTTACCAACTTGGTGGCGTTGGTT
CTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCAATACGGATTTACTTATTAATTGGCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTTACCCTATGGTTTCAATACGGATTTACTTATTAATTGG
ACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAAACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAA
AGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAATAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAAT
CTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCTCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCT
TTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGATTCAATCAAGTCAAGAGCCACACAATTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGA
TGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTGGGGGTTATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTTCTAGTGTTGAATTGGGGGTTA
AAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTGAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTG
GTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA2：oipA2:
GGCAAAAAAGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCCGGCAAAAAAGCTTTAGCAGAAAACACCTTAAATGAAGCGATCAATAACGCAAAAAATTCATTATTCCC
CGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGACGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCAACAAAGA
TCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAGCTTGGGGTATAAATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTCAATGAACTCAGCTTGGGGTATAAA
TATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGGTTACCAACTTGGTGGTATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTTCTCTTTTTTTCGGTTACCAACTTGGTGG
CGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCAATACGGATTTACTTACGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTTACCCTATGGTTTCAATACGGATTTACTTA
TTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATA
TTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATTTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAAT
CAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATC
TCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGG
AGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATT
GGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATA
AAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA3：oipA3:
TTATTCCCCGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTCTTATTCCCCGAACAAAACACAAAAGCCATAAGAGATGCGCAAAACGCCTTAAATGCAGTGAAAGATTC
AACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTCAGCTTGGAACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTCAATGAACTCAGCTTGG
GGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGGTTACCAAGGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTTCTCTTTTTTTCGGTTACCAA
CTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCAATACGGACTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTACCCTATGGTTTCAATACGGA
TTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGC
TTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGTTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGG
CACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGCACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAG
TAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTCAGTTACAAGGGAAATTTGGCG
TTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGT
GTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATT
GGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA4：oipA4:
AAAGATTCAACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACTAAAGATTCAACAAAGATCGCTAACCGATTCGCAGGAAATGGCGGGTCGGGCGGTATTTTCAATGAACT
CAGCTTGGGGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCGCAGCTTGGGGTATAAATATTTTTTGGGTAAAAAAGGGATTATAGGGTTTAGGCACTCTCTTTTTTTCG
GTTACCAACTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTCGTTACCAACTTGGTGGCGTTGGTTCTGTTCCTGGCAGCGGTTTAATAGCTTTTTTACCCTATGGTTTC
AATACGGATTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGTAATACGGATTTACTTATTAATTGGACTAACGATAAACGAGCGTCCCAAGAATATGTTGAACGAAGGGT
AAAAGGGCTTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAGAAAAGGGCTTTCTATATTTTACAAAGATATGACCGGCAGAACGCTAGACGCTAACACATTAAAAAAAAG
CATCAAGGCACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGGCATCAAGGCACATAATCAGAAAATCTTCAGGACTTGTAATTGGCATGGATATAGGGGCTAGCACTTGG
TTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAATTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTCAAGAGCCACACAATTTTTCAGTTACAAGGGAA
ATTTGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGATTTGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGG
GTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGG
GATAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACAGATAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACA
TTAATTAA
oipA5：oipA5:
CGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAAAGATATGACCGGCGAGCGTCCCAAGAATATGTTGAACGAAGGGTAAAAGGGCTTTCTATATTTTACAAAGATATGACCGG
CAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAATCTTCAGGACTTGCAGAACGCTAGACGCTAACACATTAAAAAAAGCATCAAGGCACATAATCAGAAAATCTTCAGGACTTG
TAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTCTAATTGGCATGGATATAGGGGCTAGCACTTGGTTTGCAAGTAACAATCTCACCCCTTTCAATCAAGTC
AAGAGCCACACAATTTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGATGAATACGATATAAGAGGCCACACAATTTTCAGTTACAAGGGAAATTTGGCGTTCGTTGGAGCGGTGATGAATACGATAT
TGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCTAGTGTTGAATTGGGGGTTAAAGTACCAGCGT
TTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTGGTGAGCGTTTATTTAAAGTCAATTACTATGGCGATGATTATGGGGATAAATTGGATTATAAAAGAGTGGTGAGCGTTTAT
CTCAACTATACATATAACTTTAAAAACAAACATTAACTCAACTATACATATAACTTTAAAAAACAAACATTAA
oipA6：oipA6:
GGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTCGGCGTTCGTTGGAGCGGTGATGAATACGATATTGATCGCTATGGCGATGAAATCTATCTTGGGGGTTC
TAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATATAGTGTTGAATTGGGGGTTAAAGTACCAGCGTTTAAAGTCAATTACTATGGCGATGATTATGGGGATA
AATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAACAAACATTAAAATTGGATTATAAAAGAGTGGTGAGCGTTTATCTCAACTATACATATAACTTTAAAAAACAAACATTAA
2.幽门螺杆菌oipA基因片段的表达及产物纯化2. Expression of Helicobacter pylori oipA gene fragment and product purification
2.1蛋白表达：六个oipA基因片断表达后的预测大小分别为：32KD；30KD；27KD；25KD；16KD；7.5KD，含GST以及His的融合蛋白肽段预测大小分别约为67KD；65KD；62KD；60KD；51KD；42KD。经IPTG诱导后，SDS-PAGE电泳可见，各重组菌均有一条表达增加的条带，分子量与预期的融合蛋白一致；经图像分析系统测定，表达量分别占菌体总蛋白的30.10％；30.22％；30.01％；30.48％；30.58％；44.44％，如图3。表达的融合蛋白分别命名为OipA1；OipA2；OipA3；OipA4；OipA5；OipA6，空载体转化菌蛋白命名为OipAK。2.1 Protein expression: The predicted sizes of the six oipA gene fragments after expression are: 32KD; 30KD; 27KD; 25KD; 16KD; 7.5KD. The predicted sizes of the fusion protein peptides containing GST and His are about 67KD; 65KD; 62KD; 60KD; 51KD; 42KD. After being induced by IPTG, SDS-PAGE electrophoresis showed that each recombinant bacteria had a band with increased expression, and the molecular weight was consistent with the expected fusion protein; as determined by the image analysis system, the expression levels accounted for 30.10% of the total bacterial protein; 30.22 %; 30.01%; 30.48%; 30.58%; 44.44%, as shown in Figure 3. The expressed fusion proteins were named OipA1; OipA2; OipA3; OipA4; OipA5; OipA6, and the protein transformed with empty vector was named OipAK.
2.2表达产物鉴定2.2 Identification of expression products
重组蛋白肽段经SDS-PAGE电泳并转膜后，经过Western-Breeze化学发光的X光片上相应位置各有一条特异性条带，可见各蛋白肽段均可以被GST单克隆抗体识别，证明所表达的乃是预期的融合蛋白肽段。如图4。After the recombinant protein peptides were electrophoresed by SDS-PAGE and transferred to the membrane, there was a specific band at the corresponding position on the X-ray film after Western-Breeze chemiluminescence. It can be seen that each protein peptide can be recognized by the GST monoclonal antibody, proving that the What is expressed is the expected fusion protein peptide. Figure 4.
2.3表达条件的优化2.3 Optimization of expression conditions
终浓度0.5、1、1.5、2mmol/L的IPTG均能诱导重组蛋白的表达，SDS-PAGE电泳分析显示，当IPTG终浓度为1.5mmol/L时，表达量最大。分别诱导0.5、1、2、3、4、5、6h，重组蛋白均有表达。以OipA6为例，SDS-PAGE电泳后图像分析显示，表达量分别占菌体总蛋白41.08％；46.8％；49.3％；52.47％；54.33％；47.74％和46.87％。其中，以诱导4h，含量54.33％为最高。即当诱导时间为4h时，重组蛋白的表达量最大。如图5。IPTG with a final concentration of 0.5, 1, 1.5, and 2mmol/L could induce the expression of the recombinant protein. SDS-PAGE electrophoresis analysis showed that the expression was the largest when the final concentration of IPTG was 1.5mmol/L. After induction for 0.5, 1, 2, 3, 4, 5, and 6 hours, the recombinant protein was expressed. Taking OipA6 as an example, image analysis after SDS-PAGE electrophoresis showed that the expression levels accounted for 41.08%; 46.8%; 49.3%; 52.47%; 54.33%; 47.74% and 46.87% of the total bacterial protein respectively. Among them, the content of 54.33% was the highest after 4 hours of induction. That is, when the induction time is 4 hours, the expression of the recombinant protein is the largest. Figure 5.
2.4重组蛋白肽段的纯化2.4 Purification of recombinant protein peptides
包涵体的粗纯Crude purity of inclusion bodies
对重组蛋白包涵体进行初步纯化，SDS-PAGE电泳分析显示，当尿素浓度为8mol/L时，包涵体的溶解度最大，各蛋白肽段的纯度分别达到80％；82％；84％；83％；81％；90％。如图6。Preliminary purification of the recombinant protein inclusion body, SDS-PAGE electrophoresis analysis showed that when the urea concentration was 8mol/L, the solubility of the inclusion body was the largest, and the purity of each protein peptide reached 80%; 82%; 84%; 83% ; 81%; 90%. Figure 6.
亲和纯化(His-Tag)Affinity purification (His-Tag)
经初步纯化的包涵体进行亲和层析纯化，分别洗脱4次后，SDS-PAGE电泳分析显示，纯度分别可以达到：93％-96％，如图7。The preliminarily purified inclusion bodies were purified by affinity chromatography and eluted 4 times respectively. SDS-PAGE electrophoresis analysis showed that the purity could reach 93%-96%, respectively, as shown in FIG. 7 .
2.5重组蛋白肽段浓度测定2.5 Determination of recombinant protein peptide concentration
用Bradford方法测得的重组蛋白肽段的浓度见表1。The concentrations of recombinant protein peptides measured by the Bradford method are shown in Table 1.
表1：重组蛋白浓度Table 1: Recombinant Protein Concentrations
2.6重组蛋白肽段的抗原性2.6 Antigenicity of recombinant protein peptides
Western blot结果显示，NC膜上各有一条特异性条带，条带位置与重组蛋白肽段位置一致。说明重组蛋白肽段可以被羊抗Hp多克隆抗体识别，具有抗原性。The results of Western blot showed that there was a specific band on each NC membrane, and the position of the band was consistent with the position of the recombinant protein peptide. It shows that the recombinant protein peptide can be recognized by goat anti-Hp polyclonal antibody and has antigenicity.
如图8。Figure 8.
2.7筛选抗原性强的重组蛋白肽段2.7 Screening of recombinant protein peptides with strong antigenicity
间接ELISA法检测重组蛋白肽段的抗原性，结果见表2。阳性对照Hp NCTC11637蛋白的吸光度最大，阴性对照大肠杆菌BL-21蛋白的吸光度最小，重组蛋白的吸光度从大到小依次为OipA6＞OipA5＞OipA4＞OipA3＞OipA2＞OipA1。表明同样条件下，OipA6的抗原性最强。The antigenicity of the recombinant protein peptide was detected by indirect ELISA, and the results are shown in Table 2. The positive control Hp NCTC11637 protein had the largest absorbance, the negative control Escherichia coli BL-21 protein had the smallest absorbance, and the absorbance of the recombinant protein in descending order was OipA6>OipA5>OipA4>OipA3>OipA2>OipA1. It shows that under the same conditions, OipA6 has the strongest antigenicity.
表2：重组蛋白ELISA的吸光度Table 2: Absorbance of recombinant protein ELISA
3.OipA6重组抗原检测血清相应抗体及其评价3. OipA6 recombinant antigen detection serum corresponding antibody and its evaluation
3.1临床标本分离oipA基因信号区序列分析3.1 Sequence analysis of signal region of oipA gene isolated from clinical specimens
临床标本分离培养获得83株Hp，PCR扩增其oipA信号区基因，66株细菌获得阳性结果。将66株细菌的oipA基因PCR扩增产物进行序列分析，根据信号区AG(CT)重复数目的多少，判断oipA基因的开关状态：当信号区有6、9、(1+3)、(2+3)、(1+2)、(1+1+1)、(1+1+2)个CT双核苷酸重复时，基因为“开放”状态，可编码OipA蛋白，使机体产生相应的抗体；当信号区有5个或7个CT双核苷酸重复时，基因为“关闭”状态，不编码OipA蛋白。依此判断在66株细菌中，基因处于开放状态的有62株；基因处于关闭状态的0株；无法判断开关状态的有4株。相应信号区的CT(AG)重复数目、序列、基因的开关状态如表3。83 strains of Hp were isolated and cultured from clinical specimens, and the oipA signal region gene was amplified by PCR, and 66 strains of bacteria obtained positive results. Sequence analysis was carried out on the oipA gene PCR amplification products of 66 strains of bacteria, and the switch state of the oipA gene was judged according to the number of repeats in the signal region AG (CT): when the signal region had 6, 9, (1+3), (2 When +3), (1+2), (1+1+1), (1+1+2) CT dinucleotide repeats, the gene is in the "open" state, which can encode OipA protein, so that the body can produce corresponding Antibody; when there are 5 or 7 CT dinucleotide repeats in the signal region, the gene is in the "off" state and does not encode OipA protein. According to this, among the 66 strains of bacteria, 62 strains had the gene in the open state; 0 strains had the gene in the closed state; and 4 strains could not determine the state of the switch. Table 3 shows the CT(AG) repeat number, sequence, and gene switch status of the corresponding signal region.
表3：66株Hp信号区AG(CT)重复序列分析结果Table 3: Analysis results of AG (CT) repeat sequence in the signal region of 66 strains of Hp
3.2抗原检测OipA抗体的敏感性和特异性3.2 Sensitivity and specificity of antigen detection OipA antibody
检测62例oipA信号区基因为开放状态(即可编码OipA，机体可产生抗体)的血清标本，及信号区PCR结果阴性和未感染Hp(Hp培养、血清CagA抗体、胃粘膜快速尿素酶检测同时阴性)的48例(即功能性oipA阴性)血清标本的OipA抗体，比较血清OipA抗体与oipA基因检测结果，如表4，结果显示，以OipA6作为抗原检测OipA抗体的敏感性为95.16％(59/62)，特异性为95.83％(46/48)，约登指数(Youden′s index)＝90.99％。Detect 62 cases of serum samples whose oipA signal region gene is in an open state (it can encode OipA, and the body can produce antibodies), and the PCR results of the signal region are negative and uninfected Hp (Hp culture, serum CagA antibody, gastric mucosa rapid urease detection at the same time Negative) of the OipA antibody of 48 cases (i.e. functional oipA negative) serum specimens, compare serum OipA antibody and oipA gene detection results, as shown in Table 4, the results show that the sensitivity of OipA antibody detection with OipA6 as antigen is 95.16% (59 /62), specificity was 95.83% (46/48), Youden's index (Youden's index) = 90.99%.
表4：OipA抗体检测与oipA基因检测结果比较Table 4: Comparison of OipA antibody detection and oipA gene detection results
SEQUENCE LISTINGSEQUENCE LISTING
<110>福建医科大学<110> Fujian Medical University
<120>幽门螺杆菌高毒力株检测试剂的制备<120>Preparation of detection reagents for highly virulent strains of Helicobacter pylori
<160>1<160>1
<170>PatentIn version 3.1<170>PatentIn version 3.1
<210>1<210>1
<211>203<211>203
<212>DNA<212>DNA
<213>Helicobacter pylori<213>Helicobacter pylori
<400>1<400>1
GGC GTT CGT TGG AGC GGT GAT GAA TAC GAT ATT GAT CGC TAT GGC GAT GAA ATC 54GGC GTT CGT TGG AGC GGT GAT GAA TAC GAT ATT GAT CGC TAT GGC GAT GAA ATC 54
Gly Val Arg Trp Ser Gly Asp Glu Tyr Asp Ile Asp Arg Tyr Gly Asp Glu IleGly Val Arg Trp Ser Gly Asp Glu Tyr Asp Ile Asp Arg Tyr Gly Asp Glu Ile
1 5 10 151 5 10 15
TAT CTT GGG GGT TCT AGT GTT GAA TTG GGG GTT AAA GTA CCA GCG TTT AAA GTC 108TAT CTT GGG GGT TCT AGT GTT GAA TTG GGG GTT AAA GTA CCA GCG TTT AAA GTC 108
Tyr Leu Gly Gly Ser Ser Val Glu Leu Gly Val Lys Val Pro Ala Phe Lys ValTyr Leu Gly Gly Ser Ser Val Glu Leu Gly Val Lys Val Pro Ala Phe Lys Val
20 25 30 3520 25 30 35
AAT TAC TAT GGC GAT GAT TAT GGG GAT AAA TTG GAT TAT AAA AGA GTG GTG AGC 162AAT TAC TAT GGC GAT GAT TAT GGG GAT AAA TTG GAT TAT AAA AGA GTG GTG AGC 162
Asn Tyr Tyr Gly Asp Asp Tyr Gly Asp Lys Leu Asp Tyr Lys Arg Val Val SerAsn Tyr Tyr Gly Asp Asp Tyr Gly Asp Lys Leu Asp Tyr Lys Arg Val Val Ser
40 45 5040 45 50
GTT TAT CTC AAC TAT ACA TAT AAC TTT AAA AAC AAA CAT TAA 204GTT TAT CTC AAC TAT ACA TAT AAC TTT AAA AAC AAA CAT TAA 204
Val Tyr Leu Asn Tyr Thr Tyr Asn Phe Lys Asn Lys HisVal Tyr Leu Asn Tyr Thr Tyr Asn Phe Lys Asn Lys His
55 60 6555 60 65
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636641A (en) * | 2012-03-26 | 2012-08-15 | 上海凯创生物技术有限公司 | Detection kit of helicobacter pylori emulsion method and preparation process thereof |
| CN115267208A (en) * | 2022-09-27 | 2022-11-01 | 上海芯超生物科技有限公司 | Antigen and kit for detecting helicobacter pylori antibody and preparation method thereof |
| CN116375823A (en) * | 2022-12-09 | 2023-07-04 | 扬州大学 | B cell epitope target antigen carrying virulence factors of Helicobacter pylori and its expression vector and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102636641A (en) * | 2012-03-26 | 2012-08-15 | 上海凯创生物技术有限公司 | Detection kit of helicobacter pylori emulsion method and preparation process thereof |
| CN115267208A (en) * | 2022-09-27 | 2022-11-01 | 上海芯超生物科技有限公司 | Antigen and kit for detecting helicobacter pylori antibody and preparation method thereof |
| CN116375823A (en) * | 2022-12-09 | 2023-07-04 | 扬州大学 | B cell epitope target antigen carrying virulence factors of Helicobacter pylori and its expression vector and application |
| CN116375823B (en) * | 2022-12-09 | 2024-02-23 | 扬州大学 | B cell epitope target antigen carrying helicobacter pylori virulence factor, expression vector and application thereof |
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