CN108794584A - A kind of Actinobacillus pleuropneumoniae immune protective antigen albumin A PJL_1380 and its application - Google Patents
A kind of Actinobacillus pleuropneumoniae immune protective antigen albumin A PJL_1380 and its application Download PDFInfo
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Abstract
本发明公开了一种胸膜肺炎放线杆菌免疫保护性抗原蛋白APJL_1380及其应用。本发明胸膜肺炎放线杆菌免疫保护性抗原蛋白的氨基酸序列如SEQ ID NO.1所示,该免疫保护性抗原蛋白由273个氨基酸组成,其成熟多肽部分为25‑582位氨基酸。编码该免疫保护性抗原蛋白的核苷酸序列优选如SEQ ID NO.2所示。本发明的免疫保护性抗原蛋白具有很好的免疫原性和免疫保护作用强,其具有制备用于检测胸膜肺炎放线杆菌抗体的试剂盒的应用、制备猪胸膜肺炎亚单位疫苗的应用、制备用于预防由胸膜肺炎放线杆菌引起的疾病的药物的应用。本发明为制备猪胸膜肺炎亚单位疫苗提供了新的材料,对猪胸膜肺炎的防制具有重要意义。
The invention discloses an immunoprotective antigen protein APJL_1380 of Actinobacillus pleuropneumoniae and its application. The amino acid sequence of the immunoprotective antigenic protein of Actinobacillus pleuropneumoniae of the present invention is shown in SEQ ID NO.1. The immunoprotective antigenic protein consists of 273 amino acids, and its mature polypeptide part is 25-582 amino acids. The nucleotide sequence encoding the immunoprotective antigenic protein is preferably shown in SEQ ID NO.2. The immunoprotective antigen protein of the present invention has good immunogenicity and strong immune protection effect, and it has the application of preparing a test kit for detecting the antibody of Actinobacillus pleuropneumoniae, the application of preparing porcine pleuropneumonia subunit vaccine, and the preparation of Use of drugs for the prophylaxis of diseases caused by Actinobacillus pleuropneumoniae. The invention provides a new material for preparing porcine pleuropneumonia subunit vaccine, and has great significance for the prevention and control of porcine pleuropneumonia.
Description
技术领域technical field
本发明涉及动物传染病亚单位疫苗制备技术领域,具体涉及一种胸膜肺炎放线杆菌免疫保护性抗原蛋白APJL_1380及其应用。The invention relates to the technical field of preparation of animal infectious disease subunit vaccines, in particular to an immunoprotective antigenic protein APJL_1380 of Actinobacillus pleuropneumoniae and its application.
背景技术Background technique
胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)是一种革兰氏阴性菌小杆菌,是引起猪胸膜肺炎的病原菌。该病是一种高度接触传染的呼吸道疾病,以急性出血性、纤维素性、坏死性支气管肺炎和纤维素性胸膜炎为主要特征,自1957年Pattison等首次报道以来,该病已遍布全球多个国家和地区,严重阻碍养猪业的健康发展。Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) is a small Gram-negative bacillus, which is the pathogenic bacterium that causes swine pleuropneumonia. The disease is a highly contagious respiratory disease characterized by acute hemorrhagic, fibrinous, necrotizing bronchopneumonia and fibrinous pleurisy. Since Pattison et al. areas, seriously hindering the healthy development of the pig industry.
疫苗免疫是预防和控制猪胸膜肺炎的有效手段。猪胸膜肺炎亚单位疫苗通常是以经鉴定的胸膜肺炎放线杆菌免疫保护性抗原蛋白为基础,通过适当处理制成的疫苗制剂。亚单位疫苗使用安全,能激发动物产生高水平抗目的抗原蛋白的抗体,为动物提供一定的保护。但是目前,可被人们用来生产猪胸膜肺炎亚单位疫苗的免疫保护性抗原蛋白数量较少,很大程度上限制了猪胸膜肺炎亚单位疫苗的改进和发展。因此,发掘新的免疫保护性抗原蛋白,对开发高效猪胸膜肺炎亚单位疫苗和该病的控制具有重要意义。Vaccine immunization is an effective means to prevent and control porcine pleuropneumonia. Porcine pleuropneumonia subunit vaccines are usually based on the identified immunoprotective antigen protein of Actinobacillus pleuropneumoniae, and are prepared through appropriate processing. The subunit vaccine is safe to use and can stimulate animals to produce high levels of antibodies against the target antigen protein, providing animals with certain protection. But at present, the number of immunoprotective antigen proteins that can be used to produce porcine pleuropneumonia subunit vaccines is relatively small, which largely limits the improvement and development of porcine pleuropneumonia subunit vaccines. Therefore, discovering new immunoprotective antigenic proteins is of great significance for the development of highly effective porcine pleuropneumonia subunit vaccines and the control of the disease.
发明内容Contents of the invention
本发明的目的在于提供一种新型的胸膜肺炎放线杆菌免疫保护性抗原蛋白APJL_1380。本发明的目的还在于提供所述免疫保护性抗原蛋白APJL_1380的应用。The object of the present invention is to provide a novel APJL_1380 immunoprotective antigen protein of Actinobacillus pleuropneumoniae. The purpose of the present invention is also to provide the application of the immunoprotective antigenic protein APJL_1380.
本发明的目的通过下述技术方案实现:The object of the present invention is achieved through the following technical solutions:
一种胸膜肺炎放线杆菌免疫保护性抗原蛋白APJL_1380,其氨基酸序列如SEQ IDNO.1所示,该免疫保护性抗原蛋白由582个氨基酸组成,其成熟多肽部分为25-582位氨基酸。编码该免疫保护性抗原蛋白的核苷酸序列优选如SEQ ID NO.2所示。An Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL_1380, the amino acid sequence of which is shown in SEQ ID NO.1, the immune protective antigen protein consists of 582 amino acids, and its mature polypeptide part is 25-582 amino acids. The nucleotide sequence encoding the immunoprotective antigenic protein is preferably shown in SEQ ID NO.2.
一种重组表达载体,包含所述免疫保护性抗原蛋白成熟多肽的编码序列。A recombinant expression vector, comprising the coding sequence of the mature polypeptide of the immunoprotective antigen protein.
一种重组工程菌,包含所述重组表达载体的大肠杆菌。A recombinant engineering bacterium, comprising the Escherichia coli of the recombinant expression vector.
所述胸膜肺炎放线杆菌免疫保护性抗原的制备方法,包括以下步骤:The preparation method of the Actinobacillus pleuropneumoniae immunoprotective antigen comprises the following steps:
(1)构建包含所述免疫保护性抗原蛋白或其成熟多肽编码序列的重组表达载体;所述的重组表达载体的骨架载体优选为pGEX-KG。(1) Constructing a recombinant expression vector comprising the immunoprotective antigen protein or its mature polypeptide coding sequence; the backbone vector of the recombinant expression vector is preferably pGEX-KG.
(2)将所述重组表达载体转化到宿主细胞中;所述的宿主细胞优选为大肠杆菌;(2) transforming the recombinant expression vector into a host cell; the host cell is preferably Escherichia coli;
(3)培养所述经转化的宿主细胞并诱导其表达所述免疫保护性抗原;所述的培养、诱导的条件优选为:将经转化的宿主细胞在37℃培养至OD600为0.6-0.7时,加入IPTG至终浓度1.0mM,转至37℃继续培养3-4小时。(3) cultivating the transformed host cells and inducing them to express the immunoprotective antigen; the conditions for culturing and inducing are preferably: culturing the transformed host cells at 37°C until the OD600 is 0.6-0.7 , add IPTG to a final concentration of 1.0 mM, and transfer to 37°C to continue culturing for 3-4 hours.
(4)从所述经过诱导的宿主细胞分离和纯化所述胸膜肺炎放线杆菌免疫保护性抗原。(4) Isolating and purifying the immunoprotective antigen of Actinobacillus pleuropneumoniae from the induced host cells.
所述的胸膜肺炎放线杆菌免疫保护性抗原能够与兔抗胸膜肺炎放线杆菌多克隆抗体进行结合反应,表明其具有很好的免疫反应性,可用于样品中胸膜肺炎放线杆菌抗体水平的测定。因此,上述胸膜肺炎放线杆菌免疫保护性抗原具有制备用于检测胸膜肺炎放线杆菌抗体的试剂盒的应用,所述试剂盒基于的方法包括ELISA法、蛋白印迹法、胶体金免疫法、斑点杂交法等。The described Actinobacillus pleuropneumoniae immunoprotective antigen can be combined with the rabbit anti-Actinobacillus pleuropneumoniae polyclonal antibody, indicating that it has good immunoreactivity and can be used to measure the antibody level of Actinobacillus pleuropneumoniae in the sample. Determination. Therefore, the above-mentioned Actinobacillus pleuropneumoniae immunoprotective antigen has the application of preparing a test kit for detecting the Actinobacillus pleuropneumoniae antibody. Hybridization etc.
所述的胸膜肺炎放线杆菌免疫保护性抗原免疫小鼠后,能为小鼠感染胸膜肺炎放线杆菌提供有效的免疫保护力,表明其具有很好的免疫原性和免疫保护作用。因此,所述胸膜肺炎放线杆菌免疫保护性抗原具有制备猪胸膜肺炎亚单位疫苗的应用,具有制备用于预防由胸膜肺炎放线杆菌引起的疾病的药物的应用。After the mice are immunized with the immunoprotective antigen of Actinobacillus pleuropneumoniae, it can provide effective immune protection for mice infected with Actinobacillus pleuropneumoniae, indicating that it has good immunogenicity and immune protection. Therefore, the immunoprotective antigen of Actinobacillus pleuropneumoniae has the application of preparing porcine pleuropneumonia subunit vaccine and the application of preparing medicine for preventing diseases caused by Actinobacillus pleuropneumoniae.
相应的,上述编码所述免疫保护性抗原的核苷酸、表达所述免疫保护性抗原的重组表达载体或重组工程菌,也具有这些应用:制备用于检测胸膜肺炎放线杆菌抗体的试剂盒的应用,制备猪胸膜肺炎亚单位疫苗的应用,制备用于预防由胸膜肺炎放线杆菌引起的疾病的药物的应用。Correspondingly, the above-mentioned nucleotides encoding the immunoprotective antigen, recombinant expression vectors or recombinant engineering bacteria expressing the immunoprotective antigen also have these applications: preparation of a kit for detecting antibodies against Actinobacillus pleuropneumoniae The application of the invention, the application of preparing porcine pleuropneumonia subunit vaccine, and the application of preparing medicine for preventing diseases caused by Actinobacillus pleuropneumoniae.
本发明具有如下优点和有益效果:本发明的免疫保护性抗原蛋白APJL_1380的免疫原性和免疫保护作用强,用其免疫小鼠后再以6×LD50剂量的胸膜肺炎放线杆菌感染小鼠,存活率为100%;经抗APJL_1380蛋白高免血清被动免疫的小鼠,以6×LD50剂量的胸膜肺炎放线杆菌感染小鼠,存活率为100%。本发明为制备猪胸膜肺炎亚单位疫苗提供了新的材料,对猪胸膜肺炎的防制具有重要意义。The present invention has the following advantages and beneficial effects: the immunoprotective antigenic protein APJL_1380 of the present invention has strong immunogenicity and immunoprotective effect, and after immunizing mice with it, the mice are infected with Actinobacillus pleuropneumoniae at a dose of 6× LD50 , the survival rate was 100%. The mice passively immunized with anti-APJL_1380 protein hyperimmune serum were infected with 6×LD 50 dose of Actinobacillus pleuropneumoniae, and the survival rate was 100%. The invention provides a new material for preparing porcine pleuropneumonia subunit vaccine, and has great significance for the prevention and control of porcine pleuropneumonia.
附图说明Description of drawings
图1是表达载体pGEX-KG-APJL_1380的酶切鉴定图。泳道M:DL15000DNA marker;泳道1:质粒pGEX-KG-APJL_1380BamHI/HindIII酶切。Fig. 1 is an enzyme digestion identification diagram of the expression vector pGEX-KG-APJL_1380. Lane M: DL15000 DNA marker; Lane 1: digestion of plasmid pGEX-KG-APJL_1380BamHI/HindIII.
图2是工程菌表达目的蛋白APJL_1380的SDS-PAGE检测图。泳道M:预染的蛋白质marker;泳道1:大肠杆菌/pGEX-KG-APJL_1380全菌裂解液;泳道2:大肠杆菌/pGEX-KG-APJL_1380菌体裂解上清;泳道3:大肠杆菌/pGEX-KG-APJL_1380菌体裂解沉淀;泳道4:大肠杆菌/pGEX-KG空载体全菌裂解液。Fig. 2 is the SDS-PAGE detection chart of engineering bacteria expressing the target protein APJL_1380. Lane M: Pre-stained protein marker; Lane 1: Escherichia coli/pGEX-KG-APJL_1380 whole cell lysate; Lane 2: Escherichia coli/pGEX-KG-APJL_1380 cell lysate supernatant; Lane 3: Escherichia coli/pGEX- KG-APJL_1380 cell lysate precipitate; lane 4: Escherichia coli/pGEX-KG empty vector whole bacterial lysate.
图3是免疫印迹分析目的蛋白的结果图。A图为APJL_1380纯化后的SDS-PAGE图,B图为转膜后免疫印迹图。泳道M:预染的蛋白质marker;泳道1:纯化的GST蛋白;泳道2:纯化的APJL_1380蛋白。箭头所示为APJL_1380蛋白免疫印迹显色条带。Fig. 3 is a graph showing the results of western blot analysis of the target protein. Figure A is the SDS-PAGE image of APJL_1380 after purification, and Figure B is the Western blot image after transfer. Lane M: pre-stained protein marker; Lane 1: purified GST protein; Lane 2: purified APJL_1380 protein. Arrows show APJL_1380 protein immunoblotting bands.
图4是APJL_1380蛋白的免疫保护力结果图。A图为APJL_1380免疫后攻毒小鼠存活率图,B图为抗APJL_1380多克隆抗体被动免疫小鼠攻毒后小鼠存活率图。感染后72h至观察期结束,小鼠存活率未发生变化。Fig. 4 is a graph showing the immune protection of APJL_1380 protein. Figure A is the survival rate of challenged mice after APJL_1380 immunization, and Figure B is the survival rate of mice after passive immunization with anti-APJL_1380 polyclonal antibody. From 72 hours after infection to the end of the observation period, the survival rate of the mice did not change.
具体实施方式Detailed ways
以下实施例用于进一步说明本发明,但不应理解为对本发明的限制。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
实施例1APJL_1380蛋白的表达Expression of embodiment 1 APJL_1380 protein
一、胸膜肺炎放线杆菌基因组DNA的提取1. Genomic DNA extraction of Actinobacillus pleuropneumoniae
取1mL过夜培养的胸膜肺炎放线杆菌(JL03)的菌液,12000rpm离心1分钟,弃去上清,然后按照基因组提取试剂盒(武汉博越生物技术有限公司)说明书提取JL03菌株的基因组。具体流程如下:在菌体沉淀中加入200μL RB重悬,10000rpm离心30秒,弃上清,再加入200μL RB重悬沉淀,然后,在离心管中加入20μL溶菌酶剧烈震荡,并放于37℃温箱中温育15分钟,之后再加入200μL结合液CB,颠倒混匀后加入20μL蛋白酶K(20mg/mL),混匀,放于70℃水浴锅中反应10分钟,拿出冷却后加入100μL异丙醇,颠倒混匀,然后将离心管中的所有物质转入吸附柱中,10000rpm离心30秒,倒掉废液,再加入500μL IR,12000rpm离心30秒,弃废液,接着加入700μL WB,12000rpm离心30秒,弃掉废液,再加入500μL WB,12000rpm离心30秒,弃废液,然后13000rpm离心2分钟,取出吸附柱,放入新的离心管中,在吸附膜的中间部位加50μL EB,室温放置5分钟,12000rpm离心1分钟,所得溶液即基因组,放入-20℃储存备用。Take 1 mL of overnight cultured Actinobacillus pleuropneumoniae (JL03), centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and then extract the genome of the JL03 strain according to the instructions of the genome extraction kit (Wuhan Boyue Biotechnology Co., Ltd.). The specific process is as follows: Add 200 μL RB to the bacterial pellet to resuspend, centrifuge at 10,000 rpm for 30 seconds, discard the supernatant, then add 200 μL RB to resuspend the pellet, then add 20 μL lysozyme to the centrifuge tube for vigorous shaking, and place at 37°C Incubate in the incubator for 15 minutes, then add 200 μL of binding solution CB, mix upside down, add 20 μL proteinase K (20 mg/mL), mix well, put it in a 70°C water bath for 10 minutes, take it out and add 100 μL iso Propanol, mix well by inversion, then transfer all the substances in the centrifuge tube to the adsorption column, centrifuge at 10000rpm for 30 seconds, discard the waste liquid, then add 500μL IR, centrifuge at 12000rpm for 30 seconds, discard the waste liquid, then add 700μL WB, Centrifuge at 12000rpm for 30 seconds, discard the waste liquid, then add 500μL WB, centrifuge at 12000rpm for 30 seconds, discard the waste liquid, then centrifuge at 13000rpm for 2 minutes, take out the adsorption column, put it into a new centrifuge tube, add 50μL to the middle part of the adsorption membrane EB, placed at room temperature for 5 minutes, centrifuged at 12,000 rpm for 1 minute, the resulting solution is the genome, and stored at -20°C for later use.
二、APJL_1380基因的制备2. Preparation of APJL_1380 gene
根据APJL_1380基因的核苷酸序列(如序列表的SEQID NO.2所示),设计如下引物:According to the nucleotide sequence of the APJL_1380 gene (as shown in SEQID NO.2 of the sequence listing), the following primers are designed:
正向引物:5’-TTGGATCCTGTACCGGTACAAGTTTTTTTG-3’,Forward primer: 5'-TT GGATCC TGTACCGGTACAAGTTTTTTG-3',
反向引物:5’-GGAAGCTTTTAGTTAGCGTTTTCCACTG-3’。Reverse primer: 5'-GG AAGCTTTTAGTTAGCGTTTTCCACTG -3'.
正向引物的下划线部分为BamHI酶切位点,反向引物的下划线部分为HindIII酶切位点。以胸膜肺炎放线杆菌JL03基因组DNA为模板,用设计的引物进行PCR扩增,获得APJL_1380成熟多肽编码序列。扩增体系如下:The underlined part of the forward primer is the BamHI restriction site, and the underlined part of the reverse primer is the HindIII restriction site. Genomic DNA of Actinobacillus pleuropneumoniae JL03 was used as a template, and the designed primers were used for PCR amplification to obtain the mature polypeptide coding sequence of APJL_1380. The amplification system is as follows:
PCR反应条件为:预变性:94℃5分钟;循环:94℃30秒,53℃30秒,72℃1.5分钟,30个循环;最后延伸:72℃10分钟;16℃2分钟。The PCR reaction conditions were: pre-denaturation: 94°C for 5 minutes; cycle: 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 1.5 minutes, 30 cycles; final extension: 72°C for 10 minutes; 16°C for 2 minutes.
用1%琼脂糖凝胶检测PCR产物的片段大小和产量,并用DNA纯化试剂盒(武汉博越生物科技有限公司)纯化PCR产物。具体方法如下:PCR扩增产物经琼脂糖凝胶电泳分离后,将凝胶放在切胶台上,打开紫外灯,用刀片将目的片段切下,置于无菌的1.5mL离心管中,加入300μL的溶胶液,56℃水浴5分钟,期间多次震荡,待胶块完全溶化,将溶液转入吸附柱中,8000rpm离心1分钟,倒掉废液,加入700μL漂洗液,8000rpm离心1分钟,弃掉废液,一共漂洗2次,之后,12000rpm离心1分钟,将吸附柱放入新的离心管中,在吸附膜的中间部位加入30μL灭菌去离子水,65℃水浴2分钟,12000rpm离心1分钟,收集液体即是纯化后的PCR产物,即为APJL_1380成熟蛋白的编码基因片段。1% agarose gel was used to detect the fragment size and yield of the PCR product, and the PCR product was purified with a DNA purification kit (Wuhan Boyue Biotechnology Co., Ltd.). The specific method is as follows: After the PCR amplification products are separated by agarose gel electrophoresis, put the gel on the gel cutting platform, turn on the ultraviolet lamp, cut off the target fragment with a blade, and place it in a sterile 1.5mL centrifuge tube. Add 300 μL of colloid solution, bathe in 56°C water for 5 minutes, shake it several times during the period, and transfer the solution to the adsorption column after the gel block is completely dissolved, centrifuge at 8000 rpm for 1 minute, discard the waste liquid, add 700 μL of rinse solution, and centrifuge at 8000 rpm for 1 minute , discard the waste liquid, rinse twice in total, then centrifuge at 12000rpm for 1 minute, put the adsorption column into a new centrifuge tube, add 30 μL sterilized deionized water to the middle part of the adsorption membrane, bathe in 65℃ water for 2 minutes, 12000rpm Centrifuge for 1 minute, and the collected liquid is the purified PCR product, which is the coding gene fragment of APJL_1380 mature protein.
三、重组表达载体的构建3. Construction of recombinant expression vector
(1)将目的基因与A/T克隆载体pMD18-T连接:将回收到的PCR产物连接到pMD18-T载体上,连接体系为:(1) Ligate the target gene with the A/T cloning vector pMD18-T: connect the recovered PCR product to the pMD18-T vector, the connection system is:
混匀后用封口膜封口,标记为pMD18-T-APJL_1380连接产物,置16℃水浴锅中连接过夜。After mixing, it was sealed with parafilm, labeled as pMD18-T-APJL_1380 ligation product, and placed in a 16°C water bath for overnight ligation.
(2)连接产物的转化和质粒的鉴定(2) Transformation of ligation products and identification of plasmids
将pMD18-T-APJL_1380连接产物转化至大肠杆菌DH5α感受态细胞,转化方法为:取10μL连接产物加到大肠杆菌DH5α感受态细胞中,混匀后冰浴20分钟,42℃水浴热激90秒之后,迅速冰浴2分钟,然后加入500μL LB液体培养基,37℃220rpm摇床培养1小时,然后7000rpm离心3分钟,丢弃上清500μL,用剩余培养基重悬菌体,均匀涂于含有100μg/mL氨苄青霉素的LB琼脂平板上,37℃温箱培养过夜。然后挑取单菌落,接种到含有100μg/mL氨苄青霉素的LB液体培养基中,培养12小时。Transform the pMD18-T-APJL_1380 ligation product into E. coli DH5α competent cells. The transformation method is as follows: take 10 μL of the ligation product and add it to E. coli DH5α competent cells, mix well, and ice-bath for 20 minutes, then heat shock in 42°C water bath for 90 seconds After that, quickly ice-bath for 2 minutes, then add 500 μL LB liquid medium, incubate on a shaker at 37°C at 220 rpm for 1 hour, then centrifuge at 7000 rpm for 3 minutes, discard 500 μL of the supernatant, resuspend the bacteria with the remaining medium, and spread evenly on the cells containing 100 μg /mL ampicillin on LB agar plates, cultured overnight in a 37°C incubator. Then a single colony was picked and inoculated into LB liquid medium containing 100 μg/mL ampicillin, and cultured for 12 hours.
通过碱裂解法提取质粒:将菌液转入1.5mL离心管中,12000rpm离心1分钟,弃去上清。加入100μL预冷的溶液I,重悬菌体,置于冰上;加入200μL溶液II,颠倒混匀5-6次,冰浴10分钟;加入150μL预冷的溶液III,颠倒混匀5-6次,冰浴10分钟,12000rpm离心10分钟。将上清转移至新的离心管,并加入400μL异丙醇,颠倒混匀,室温放置10分钟,12000rpm离心10分钟,弃去上清,加入200μL去离子水,溶解沉淀,然后加入100μL 7.5mol/L的醋酸铵,颠倒混匀后,冰浴10分钟。12000rpm离心10分钟。然后将上清转移到一个新的离心管中,加入300μL异丙醇,颠倒混匀,室温放置10分钟。然后12000rpm离心10分钟,弃去上清,加入200μL75%乙醇,12000rpm离心2分钟,吸弃上清,加入20μL含有RNA酶的水,溶解沉淀,得到质粒。然后通过双酶切法进行鉴定,酶切体系如下:Extract the plasmid by alkaline lysis: transfer the bacterial solution into a 1.5mL centrifuge tube, centrifuge at 12000rpm for 1 minute, and discard the supernatant. Add 100 μL of pre-cooled solution I, resuspend the bacteria, and place on ice; add 200 μL of solution II, invert and mix 5-6 times, and ice-bath for 10 minutes; add 150 μL of pre-cooled solution III, invert and mix for 5-6 time, ice bath for 10 minutes, and centrifuge at 12000rpm for 10 minutes. Transfer the supernatant to a new centrifuge tube, add 400 μL of isopropanol, mix by inversion, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 200 μL of deionized water to dissolve the precipitate, and then add 100 μL of 7.5 mol /L of ammonium acetate, mixed by inversion, ice bath for 10 minutes. Centrifuge at 12000 rpm for 10 minutes. Then transfer the supernatant to a new centrifuge tube, add 300 μL isopropanol, mix by inverting, and place at room temperature for 10 minutes. Then centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 200 μL of 75% ethanol, centrifuge at 12,000 rpm for 2 minutes, aspirate and discard the supernatant, add 20 μL of water containing RNase, dissolve the precipitate, and obtain a plasmid. Then it is identified by the double enzyme digestion method. The enzyme digestion system is as follows:
37℃反应1小时后,加入终止液,混匀后取5μL样品,经1%琼脂糖凝胶电泳检测,将含有正确插入片段的质粒命名为pMD18-T-APJL_1380。通过双向测序分析目的基因序列的正确性。After reacting at 37°C for 1 hour, add stop solution, mix well, take 5 μL sample, detect by 1% agarose gel electrophoresis, and designate the plasmid containing the correct insert fragment as pMD18-T-APJL_1380. The correctness of the target gene sequence was analyzed by bidirectional sequencing.
(3)重组表达质粒的构建与鉴定(3) Construction and identification of recombinant expression plasmids
使用BamHI和HindIII双酶切质粒pMD18-T-APJL_1380,经琼脂糖凝胶电泳分离后,回收目的片段,并与经过同样酶切处理的原核表达质粒pGEX-KG连接,连接反应体系如下:The plasmid pMD18-T-APJL_1380 was double digested with BamHI and HindIII, separated by agarose gel electrophoresis, the target fragment was recovered, and ligated with the prokaryotic expression plasmid pGEX-KG that had undergone the same digestion treatment. The ligation reaction system was as follows:
混匀后用封口膜封口,标记为pGEX-KG-APJL_1380,连接产物16℃反应过夜后,将连接产物转化至大肠杆菌DH5α感受态细胞,转化方法、单菌落挑取培养、质粒制备方法同上述pMD18-T-APJL_1380质粒的构建。用BamHI和HindIII双酶切质粒pGEX-KG-APJL_1380之后,得到目的片段和载体大小与预期相符(图1)。After mixing, seal with parafilm and mark it as pGEX-KG-APJL_1380. After the ligation product was reacted at 16°C overnight, the ligation product was transformed into E. coli DH5α competent cells. The transformation method, single colony picking culture, and plasmid preparation method were the same as above. Construction of pMD18-T-APJL_1380 plasmid. After the plasmid pGEX-KG-APJL_1380 was double digested with BamHI and HindIII, the target fragment and the size of the vector were obtained as expected (Figure 1).
四、工程菌株的制备4. Preparation of engineering strains
将鉴定正确的pGEX-KG-APJL_1380质粒转化至大肠杆菌表达菌株BL21(DE3)感受态细胞,涂布含有100μg/mL氨苄青霉素的LB琼脂平板,37℃过夜培养后,挑取单菌落,37℃培养12小时,得到含有pGEX-KG-APJL_1380的重组菌,即为工程菌。同时,用pGEX-KG载体代替pGEX-KG-APJL_1380质粒,步骤同上,得到含有pGEX-KG的重组菌,用作为对照。Transform the correctly identified pGEX-KG-APJL_1380 plasmid into Escherichia coli expression strain BL21(DE3) competent cells, smear the LB agar plate containing 100 μg/mL ampicillin, and culture overnight at 37°C, pick a single colony, and 37°C Cultivate for 12 hours to obtain the recombinant bacteria containing pGEX-KG-APJL_1380, which is the engineering bacteria. At the same time, the pGEX-KG vector was used to replace the pGEX-KG-APJL_1380 plasmid, and the procedure was the same as above to obtain a recombinant bacterium containing pGEX-KG, which was used as a control.
五、APJL_1380蛋白的制备和纯化5. Preparation and purification of APJL_1380 protein
(1)将以上“四”中制备的工程菌培养于100mL含有100μg/mL氨苄青霉素的LB液体培养基,37℃培养3小时,至OD600=0.6时,加入IPTG至终浓度为1.0mM,37℃继续培养3小时。(1) Cultivate the engineered bacteria prepared in "4" above in 100 mL of LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C for 3 hours until OD 600 =0.6, add IPTG to a final concentration of 1.0 mM, Incubation was continued at 37°C for 3 hours.
(2)5000rpm离心10分钟收集菌体,将所得菌体用20mL PBS溶液重悬,用高压破碎仪破碎菌体,取样之后12000rpm离心10分钟,分别收集上清和沉淀,取样经SDS-PAGE检测APJL_1380蛋白的表达情况。如图2,SDS-PAGE电泳显示APJL_1380蛋白分子量约为53kDa(包含GST标签),GST对照蛋白分子量约为26kDa,APJL_1380主要在细菌裂解上清中。然后,将上清加入到谷胱甘肽亲和层析柱,结合完毕后,加20mL PBS洗涤亲和层析柱,然后加入5mL还原型谷胱甘肽溶液洗脱目的蛋白,并将洗脱液收集于1.5mL洁净离心管中,得到纯化的重组APJL_1380蛋白。再依次加入20mL洗脱液、20mL PBS洗涤层析柱,最后用10mL 20%乙醇浸泡保存层析柱。(2) Collect the bacteria by centrifugation at 5000rpm for 10 minutes, resuspend the obtained bacteria with 20mL PBS solution, crush the bacteria with a high-pressure crusher, and centrifuge at 12000rpm for 10 minutes after sampling, collect the supernatant and precipitate respectively, and take samples to detect APJL_1380 by SDS-PAGE protein expression. As shown in Figure 2, SDS-PAGE electrophoresis shows that the molecular weight of APJL_1380 protein is about 53kDa (including GST tag), the molecular weight of GST control protein is about 26kDa, and APJL_1380 is mainly in the bacterial lysate supernatant. Then, add the supernatant to the glutathione affinity chromatography column, after the binding is completed, add 20mL PBS to wash the affinity chromatography column, then add 5mL reduced glutathione solution to elute the target protein, and the elution The liquid was collected in a 1.5mL clean centrifuge tube to obtain purified recombinant APJL_1380 protein. Then add 20mL of eluent and 20mL of PBS to wash the chromatography column, and finally soak and preserve the chromatography column with 10mL of 20% ethanol.
(3)按上述方法从含有pGEX-KG的重组菌中提取并纯化对照蛋白GST,并通过SDS-PAGE检测纯化的APJL_1380和GST蛋白(图3A)。(3) The control protein GST was extracted and purified from the recombinant bacteria containing pGEX-KG according to the above method, and the purified APJL_1380 and GST proteins were detected by SDS-PAGE (Fig. 3A).
六、APJL_1380蛋白的免疫印迹法鉴定6. Identification of APJL_1380 protein by immunoblotting
对以上“五”中纯化的重组APJL_1380蛋白进行免疫印迹法分析,方法如下:首先,制备12%的聚丙烯酰胺凝胶,将APJL_1380和GST蛋白点样,进行SDS-PAGE电泳分离。电泳结束后,测量需要转膜的凝胶大小,然后裁剪4张等大的滤纸和1张稍大的硝酸纤维素滤膜,按照:黑色夹板-海绵垫-两层滤纸-凝胶-滤膜-两层滤纸-海绵垫-白色夹板的顺序,安装转膜装置,每铺一层,用玻璃棒赶走气泡;接通电源,80V恒压转印30分钟。转印结束后将硝酸纤维素滤膜取下,用含5%脱脂牛奶的封闭液常温封闭1小时;取出滤膜,用洗涤液洗涤3次,每次5分钟;然后加入用洗涤液以1:400稀释的兔抗胸膜肺炎放线杆菌多克隆抗体,37℃孵育30分钟;取出滤膜,用洗涤液洗涤4次,每次5分钟;然后加入1:5000稀释的HRP标记的羊抗兔IgG,37℃孵育30分钟;取出滤膜,用洗涤液洗涤5次,每次5分钟;然后用DAB显色试剂盒显色。结果表明,APJL_1380蛋白泳道出现与预期一致的显色条带,而GST蛋白没有出现条带,如图3B所示。The recombinant APJL_1380 protein purified in the above "five" was analyzed by immunoblotting, and the method was as follows: first, a 12% polyacrylamide gel was prepared, APJL_1380 and GST protein were spotted, and separated by SDS-PAGE electrophoresis. After electrophoresis, measure the size of the gel that needs to be transferred to the membrane, and then cut 4 pieces of filter paper of equal size and 1 piece of slightly larger nitrocellulose filter membrane, according to: black splint-sponge pad-two layers of filter paper-gel-filter membrane -In the order of two layers of filter paper-sponge pad-white splint, install the transfer film device, and use a glass rod to drive away the air bubbles for each layer; turn on the power supply, and transfer at a constant voltage of 80V for 30 minutes. After transfer, remove the nitrocellulose filter membrane and block it with blocking solution containing 5% skimmed milk at room temperature for 1 hour; take out the filter membrane and wash it with washing solution for 3 times, each time for 5 minutes; then add washing solution for 1 :400 diluted rabbit anti-Actinobacillus pleuropneumoniae polyclonal antibody, incubated at 37°C for 30 minutes; took out the filter membrane, washed 4 times with washing solution, 5 minutes each time; then added 1:5000 diluted HRP-labeled goat anti-rabbit For IgG, incubate at 37°C for 30 minutes; take out the filter membrane and wash it with washing solution 5 times for 5 minutes each time; then use the DAB color development kit to develop color. The results showed that the APJL_1380 protein lane had the same color band as expected, but the GST protein had no band, as shown in Figure 3B.
实施例2APJL_1380蛋白免疫保护性的鉴定Example 2 Identification of immune protection of APJL_1380 protein
(1)小鼠的主动免疫与攻毒(1) Active immunization and challenge of mice
以实施例1中纯化的APJL_1380蛋白和GST蛋白分别免疫6周龄雌性BALB/c小鼠(购于湖北省疾病预防控制中心实验动物中心),每组12只。同时设置TSB空白对照,不进行免疫。第一次免疫剂量为80μg/只,与等体积弗氏完全佐剂混合乳化,于小鼠背部皮下多点注射接种。间隔两周后,进行第二次免疫,剂量为80μg/只,与等体积弗氏不完全佐剂混合乳化,于小鼠背部皮下多点注射接种,10天后,用ELISA检测抗体水平,以HRP标记的羊抗鼠IgG为二抗(1:5000稀释)。小鼠抗APJL_1380蛋白的抗体效价达1:3.8×104。二免后14天,进行攻毒测试,以6×106CUF/只(6×LD50)剂量的血清1型胸膜肺炎放线杆菌4074株经腹腔注射感染各组小鼠,连续观察7天,记录各组小鼠的发病及死亡情况。如图4A所示,APJL_1380免疫组小鼠存活率为100%,GST免疫组及TSB空白对照组小鼠存活率为0%。Six-week-old female BALB/c mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention) were immunized with APJL_1380 protein and GST protein purified in Example 1, 12 in each group. At the same time, a TSB blank control was set without immunization. The first immunization dose was 80 μg/mouse, mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple points on the back of the mouse. After an interval of two weeks, the second immunization was carried out, the dose was 80 μg/mouse, mixed with an equal volume of Freund’s incomplete adjuvant, emulsified, and injected subcutaneously at multiple points on the back of the mouse. After 10 days, the antibody level was detected by ELISA, and HRP Labeled goat anti-mouse IgG was used as the secondary antibody (1:5000 dilution). The antibody titer of mouse anti-APJL_1380 protein reached 1:3.8×10 4 . 14 days after the second immunization, the challenge test was carried out, and the mice in each group were infected by intraperitoneal injection with 6×10 6 CUF/mouse (6×LD 50 ) dose of Serum Type 1 Actinobacillus pleuropneumoniae 4074, and the mice were continuously observed for 7 days , Record the incidence and death of mice in each group. As shown in Figure 4A, the survival rate of the mice in the APJL_1380 immunized group was 100%, and the survival rate of the mice in the GST immunized group and the TSB blank control group was 0%.
(2)小鼠的被动免疫与攻毒(2) Passive immunization and challenge of mice
在上一步主动免疫中,收集各组二次免疫后10天的小鼠血清,组内混合。然后经腹腔注射法,分别接种6周龄雌性BALB/c小鼠(购于湖北省疾病预防控制中心实验动物中心),每组12只,每只接种50μL,进行被动免疫接种。于接种后3小时,通过腹腔注射法,以6×106CUF/只剂量的血清1型胸膜肺炎放线杆菌4074株经腹腔注射感染各组小鼠,连续观察7天,记录各组小鼠的发病及死亡情况。如图4B所示,抗APJL_1380蛋白的免疫血清组小鼠存活率为100%,抗GST蛋白的免疫血清和TSB空白血清组小鼠存活率为0%。In the previous step of active immunization, the serum of mice in each group was collected 10 days after the second immunization, and mixed within the group. Then, by intraperitoneal injection, 6-week-old female BALB/c mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention) were inoculated, 12 mice in each group, and each mouse was inoculated with 50 μL for passive immunization. Three hours after inoculation, the mice in each group were infected by intraperitoneal injection with 6×10 6 CUF/mouse of Actinobacillus pleuropneumoniae 4074 strain serotype 1, observed continuously for 7 days, and the mice in each group were recorded. morbidity and mortality. As shown in Figure 4B, the survival rate of the mice in the anti-APJL_1380 protein immune serum group was 100%, and the survival rate of the mice in the anti-GST protein immune serum and TSB blank serum groups was 0%.
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.
序列表sequence listing
<110> 华中师范大学<110> Central China Normal University
<120> 一种胸膜肺炎放线杆菌免疫保护性抗原蛋白APJL_1380及其应用<120> APJL_1380, an immunoprotective antigenic protein of Actinobacillus pleuropneumoniae and its application
<160> 4<160> 4
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 582<211> 582
<212> PRT<212> PRT
<213> Actinobacillus pleuropneumoniae<213> Actinobacillus pleuropneumoniae
<400> 1<400> 1
Met Ala Thr Ile Leu Lys Gln Lys Ile Lys Thr Val Phe Val Pro ThrMet Ala Thr Ile Leu Lys Gln Lys Ile Lys Thr Val Phe Val Pro Thr
1 5 10 151 5 10 15
Ala Met Ala Leu Phe Leu Ser Ala Cys Thr Gly Thr Ser Phe Phe GluAla Met Ala Leu Phe Leu Ser Ala Cys Thr Gly Thr Ser Phe Phe Glu
20 25 30 20 25 30
Asn Pro Leu Thr Lys Thr Val Lys Asp Glu Ala Tyr Ala Thr Ser GluAsn Pro Leu Thr Lys Thr Val Lys Asp Glu Ala Tyr Ala Thr Ser Glu
35 40 45 35 40 45
Phe Tyr Ile Asn Lys Ala Asp Arg Ala Thr Asp Lys Glu Asp Lys IlePhe Tyr Ile Asn Lys Ala Asp Arg Ala Thr Asp Lys Glu Asp Lys Ile
50 55 60 50 55 60
Thr Tyr Arg Leu Leu Ala Val Arg Lys Leu Ile Asp Glu Asn Lys AlaThr Tyr Arg Leu Leu Ala Val Arg Lys Leu Ile Asp Glu Asn Lys Ala
65 70 75 8065 70 75 80
Ala Glu Ala Gln Asn Thr Phe Asp Asp Leu Thr Leu Ser Leu Ala AspAla Glu Ala Gln Asn Thr Phe Asp Asp Leu Thr Leu Ser Leu Ala Asp
85 90 95 85 90 95
Ile Gln Lys Asn Glu Ile Gln Lys Val Glu Tyr Asn Leu Val Ala AlaIle Gln Lys Asn Glu Ile Gln Lys Val Glu Tyr Asn Leu Val Ala Ala
100 105 110 100 105 110
Gln Leu Ala Ala Leu Gln Gly Asn Glu Ala Gln Ala Val Ser Leu LeuGln Leu Ala Ala Leu Gln Gly Asn Glu Ala Gln Ala Val Ser Leu Leu
115 120 125 115 120 125
Arg Leu Val Pro Thr Thr Gln Leu Ser Arg Thr Gln Ser Met Arg TyrArg Leu Val Pro Thr Thr Gln Leu Ser Arg Thr Gln Ser Met Arg Tyr
130 135 140 130 135 140
Tyr Gln Thr Gln Ala Arg Ile Ala Glu Asn Arg Lys Asp Val Leu GluTyr Gln Thr Gln Ala Arg Ile Ala Glu Asn Arg Lys Asp Val Leu Glu
145 150 155 160145 150 155 160
Ala Val Arg Ala Arg Ser Leu Met Thr Ser Gln Leu Ile Asp Asn LysAla Val Arg Ala Arg Ser Leu Met Thr Ser Gln Leu Ile Asp Asn Lys
165 170 175 165 170 175
Leu Arg Gln Glu Asn Asn Asn Gln Ile Trp Ser Leu Leu Arg Asn AlaLeu Arg Gln Glu Asn Asn Asn Gln Ile Trp Ser Leu Leu Arg Asn Ala
180 185 190 180 185 190
Asn Lys Gly Ala Leu Ser Ile Ala Asn Pro Gly Pro Gly Glu Thr GluAsn Lys Gly Ala Leu Ser Ile Ala Asn Pro Gly Pro Gly Glu Thr Glu
195 200 205 195 200 205
Phe Ala Gly Trp Leu Ala Leu Ile Ala Val Tyr Asn Gln Asn Val SerPhe Ala Gly Trp Leu Ala Leu Ile Ala Val Tyr Asn Gln Asn Val Ser
210 215 220 210 215 220
Thr Pro Ala Gln Met Pro Gln Gly Ile Asn Asn Trp Lys Gln Leu TyrThr Pro Ala Gln Met Pro Gln Gly Ile Asn Asn Trp Lys Gln Leu Tyr
225 230 235 240225 230 235 240
Pro Asn His Ser Ala Ile Thr Val Met Pro Ala Glu Leu Gln Asn ValPro Asn His Ser Ala Ile Thr Val Met Pro Ala Glu Leu Gln Asn Val
245 250 255 245 250 255
Ser Asn Phe Gln Gln Thr Gln Leu Asn Gly Val Ala Leu Leu Leu ProSer Asn Phe Gln Gln Thr Gln Leu Asn Gly Val Ala Leu Leu Leu Pro
260 265 270 260 265 270
Leu Ser Gly Asp Ala Lys Ile Leu Gly Asp Ile Ile Lys Lys Gly PheLeu Ser Gly Asp Ala Lys Ile Leu Gly Asp Ile Ile Lys Lys Gly Phe
275 280 285 275 280 285
Asn Asp Ala Lys Gly Ala Asp Ser Ile Pro Val Gln Thr Tyr Asp ThrAsn Asp Ala Lys Gly Ala Asp Ser Ile Pro Val Gln Thr Tyr Asp Thr
290 295 300 290 295 300
Asp Ser Gly Ser Val Glu Ser Ile Leu Ala Gln Ala Lys Gln Gln GlyAsp Ser Gly Ser Val Glu Ser Ile Leu Ala Gln Ala Lys Gln Gln Gly
305 310 315 320305 310 315 320
Ala Gln Thr Ile Ile Gly Pro Leu Leu Lys Ser Arg Val Asp Glu MetAla Gln Thr Ile Ile Gly Pro Leu Leu Lys Ser Arg Val Asp Glu Met
325 330 335 325 330 335
Leu Leu Ser Pro Glu Ile Arg Asn Val Asn Val Leu Ala Leu Asn SerLeu Leu Ser Pro Glu Ile Arg Asn Val Asn Val Leu Ala Leu Asn Ser
340 345 350 340 345 350
Thr Pro Asn Val Lys Ala Ile Pro Gly Val Cys Tyr Tyr Gly Leu SerThr Pro Asn Val Lys Ala Ile Pro Gly Val Cys Tyr Tyr Gly Leu Ser
355 360 365 355 360 365
Pro Glu Ala Glu Ala Arg Ala Gly Ala Asp Arg Leu Tyr Arg Asp GlyPro Glu Ala Glu Ala Arg Ala Gly Ala Asp Arg Leu Tyr Arg Asp Gly
370 375 380 370 375 380
Tyr Ser Arg Ala Ile Val Ala Ala Ser Gln Asp Asp Phe Gly Gln ArgTyr Ser Arg Ala Ile Val Ala Ala Ser Gln Asp Asp Phe Gly Gln Arg
385 390 395 400385 390 395 400
Ser Ala Asp Ala Phe Ser Gln Arg Trp Arg Gln Leu Thr Asn Thr AspSer Ala Asp Ala Phe Ser Gln Arg Trp Arg Gln Leu Thr Asn Thr Asp
405 410 415 405 410 415
Ala Asp Val Arg Tyr Tyr Asn Ile Pro Gln Asp Ala Val Val Ala IleAla Asp Val Arg Tyr Tyr Asn Ile Pro Gln Asp Ala Val Val Ala Ile
420 425 430 420 425 430
Gln Asn Ser Gly Gly Val Gln Gly Ala Ala Leu Tyr Ala Leu Gly ThrGln Asn Ser Gly Gly Val Gln Gly Ala Ala Leu Tyr Ala Leu Gly Thr
435 440 445 435 440 445
Ala Glu Gln Leu Leu Glu Leu Lys Gln Gly Ile Asp Gly Ser Ser LeuAla Glu Gln Leu Leu Glu Leu Lys Gln Gly Ile Asp Gly Ser Ser Ser Leu
450 455 460 450 455 460
Ala Gly Gln Leu Asn Ile Tyr Thr Ser Ser Arg Ser Asn Ser Pro AsnAla Gly Gln Leu Asn Ile Tyr Thr Ser Ser Arg Ser Asn Ser Pro Asn
465 470 475 480465 470 475 480
Asn Gly Ile Glu Phe Arg Thr Ala Met Glu Gly Val Lys Phe Ser GluAsn Gly Ile Glu Phe Arg Thr Ala Met Glu Gly Val Lys Phe Ser Glu
485 490 495 485 490 495
Ile Pro Leu Leu Ala Asp Ser Asn Ser Asp Glu Tyr Lys Lys Ala GluIle Pro Leu Leu Ala Asp Ser Asn Ser Asp Glu Tyr Lys Lys Ala Glu
500 505 510 500 505 510
Thr Leu Ala Glu Ser Asp Phe Ser Met Met Arg Leu Tyr Ala Met GlyThr Leu Ala Glu Ser Asp Phe Ser Met Met Arg Leu Tyr Ala Met Gly
515 520 525 515 520 525
Ser Asp Ala Trp Ala Leu Ala Asn Lys Phe Asn Glu Phe Arg Gln IleSer Asp Ala Trp Ala Leu Ala Asn Lys Phe Asn Glu Phe Arg Gln Ile
530 535 540 530 535 540
Pro Gly Tyr Ser Val Ser Gly Leu Thr Gly Asn Leu Thr Ala Ser ProPro Gly Tyr Ser Val Ser Gly Leu Thr Gly Asn Leu Thr Ala Ser Pro
545 550 555 560545 550 555 560
Asn Cys Asn Ile Glu Arg Gly Met Ser Trp Leu Gln Tyr Arg Asn GlyAsn Cys Asn Ile Glu Arg Gly Met Ser Trp Leu Gln Tyr Arg Asn Gly
565 570 575 565 570 575
Ala Val Glu Asn Ala AsnAla Val Glu Asn Ala Asn
580 580
<210> 2<210> 2
<211> 1749<211> 1749
<212> DNA<212>DNA
<213> Actinobacillus pleuropneumoniae<213> Actinobacillus pleuropneumoniae
<400> 2<400> 2
atggcgacta ttttaaaaca gaaaataaaa actgtctttg tcccaaccgc aatggcgctt 60atggcgacta ttttaaaaca gaaaataaaa actgtctttg tcccaaccgc aatggcgctt 60
tttctttctg cttgtaccgg tacaagtttt tttgaaaacc cattaaccaa aacggtcaaa 120tttctttctg cttgtaccgg tacaagtttt tttgaaaacc cattaaccaa aacggtcaaa 120
gacgaagcat acgcaacgtc ggagttttat atcaataaag cggatcgagc cacagataaa 180gacgaagcat acgcaacgtc ggagttttat atcaataaag cggatcgagc cacagataaa 180
gaagataaaa ttacttatcg cctattagcc gttcgtaaac tgattgatga aaataaagcg 240gaagataaaa ttacttatcg cctattagcc gttcgtaaac tgattgatga aaataaagcg 240
gcagaagcac aaaatacctt cgacgatctc actctatcgc tcgccgatat ccagaaaaat 300gcagaagcac aaaatacctt cgacgatctc actctatcgc tcgccgatat ccagaaaaat 300
gaaattcaaa aagtagaata taacttggtt gcggcgcaac ttgcggcatt acagggtaat 360gaaattcaaa aagtagaata taacttggtt gcggcgcaac ttgcggcatt acagggtaat 360
gaagcacaag ccgtttcact tttaagactg gtgccgacga cccaattaag ccgtacgcaa 420gaagcacaag ccgtttcact tttaagactg gtgccgacga cccaattaag ccgtacgcaa 420
agtatgcgtt actatcaaac acaagcgcgt attgcggaaa atcgtaaaga cgtgcttgaa 480agtatgcgtt actatcaaac acaagcgcgt attgcggaaa atcgtaaaga cgtgcttgaa 480
gcggtgagag cgcgttcttt aatgacttct caattaatcg ataataagtt acgccaagaa 540gcggtgagag cgcgttcttt aatgacttct caattaatcg ataataagtt acgccaagaa 540
aataacaatc aaatttggtc attattacgt aatgcgaata aaggtgcgtt gtctattgcc 600aataacaatc aaatttggtc attattacgt aatgcgaata aaggtgcgtt gtctattgcc 600
aatccgggac cgggcgaaac cgagtttgcc ggttggttag ctttaattgc ggtttacaac 660aatccgggac cgggcgaaac cgagtttgcc ggttggttag ctttaattgc ggtttacaac 660
caaaacgttt cgacaccggc acaaatgccg cagggtatta ataactggaa acaactctat 720caaaacgttt cgacaccggc acaaatgccg cagggtatta ataactggaa acaactctat 720
ccgaatcata gtgcgataac ggtcatgccg gcggagttac aaaacgtatc gaatttccaa 780ccgaatcata gtgcgataac ggtcatgccg gcggagttac aaaacgtatc gaatttccaa 780
caaacccaat taaacggcgt cgctttactt ttaccgttaa gcggtgacgc taaaatttta 840caaacccaat taaacggcgt cgctttactt ttaccgttaa gcggtgacgc taaaatttta 840
ggcgatatta tcaaaaaagg ctttaatgac gctaaaggcg cggactccat tccggtgcaa 900ggcgatatta tcaaaaaagg ctttaatgac gctaaaggcg cggactccat tccggtgcaa 900
acatacgata cagatagcgg ttcggtcgaa agtattttgg cacaggctaa gcaacaaggc 960acatacgata cagatagcgg ttcggtcgaa agtattttgg cacaggctaa gcaacaaggc 960
gcacaaacga ttatcggtcc gttactaaaa tctcgtgtcg atgaaatgtt gttaagtcct 1020gcacaaacga ttatcggtcc gttactaaaa tctcgtgtcg atgaaatgtt gttaagtcct 1020
gaaatccgaa atgtgaatgt attggcttta aattcgacgc caaatgtaaa agcgattccg 1080gaaatccgaa atgtgaatgt attggcttta aattcgacgc caaatgtaaa agcgattccg 1080
ggcgtatgtt attacggttt atcgccggaa gcggaagcga gagccggagc ggatcgctta 1140ggcgtatgtt attacggttt atcgccggaa gcggaagcga gagccggagc ggatcgctta 1140
tatcgcgacg gttattcgcg tgctatcgtt gccgcatcac aagatgattt cggacaacgt 1200tatcgcgacg gttattcgcg tgctatcgtt gccgcatcac aagatgattt cggacaacgt 1200
tccgcagatg cattctcaca acgttggcga caattaacca atacggatgc ggacgtacgc 1260tccgcagatg cattctcaca acgttggcga caattaacca atacggatgc ggacgtacgc 1260
tattacaata ttcctcaaga tgcggttgtc gcaattcaga attcgggcgg tgtgcaaggt 1320tattacaata ttcctcaaga tgcggttgtc gcaattcaga attcgggcgg tgtgcaaggt 1320
gcggcattat atgcgttagg taccgccgag cagttacttg aattaaaaca aggtatcgac 1380gcggcattat atgcgttagg taccgccgag cagttacttg aattaaaaca aggtatcgac 1380
ggttcatcgc ttgccggaca attgaatatt tatacctctt cacgcagtaa ttcgccgaat 1440ggttcatcgc ttgccggaca attgaatatt tatacctctt cacgcagtaa ttcgccgaat 1440
aacggtatcg aattccgtac cgcaatggaa ggagtgaaat tcagtgaaat tcctctgctt 1500aacggtatcg aattccgtac cgcaatggaa ggagtgaaat tcagtgaaat tcctctgctt 1500
gcggactcga attcggacga atataaaaaa gcggaaactt tagcggaaag tgatttctcg 1560gcggactcga attcggacga atataaaaaa gcggaaactt tagcggaaag tgatttctcg 1560
atgatgcgtt tatatgcaat gggttccgat gcttgggcgt tagcaaataa attcaatgaa 1620atgatgcgtt tatatgcaat gggttccgat gcttgggcgt tagcaaataa attcaatgaa 1620
ttccgccaaa ttccgggtta tagcgtttca ggtttaaccg gtaatttaac cgccagtcct 1680ttccgccaaa ttccgggtta tagcgtttca ggtttaaccg gtaatttaac cgccagtcct 1680
aactgtaata tcgaacgcgg aatgtcttgg ttgcaatatc gtaatggcgc agtggaaaac 1740aactgtaata tcgaacgcgg aatgtcttgg ttgcaatatc gtaatggcgc agtggaaaac 1740
gctaactaa 1749gctaactaa 1749
<210> 3<210> 3
<211> 30<211> 30
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)
<400> 3<400> 3
ttggatcctg taccggtaca agtttttttg 30ttggatcctg taccggtaca agtttttttg 30
<210> 4<210> 4
<211> 28<211> 28
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)
<400> 4<400> 4
ggaagctttt agttagcgtt ttccactg 28ggaagctttt agttagcgttttccactg 28
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112704732A (en) * | 2020-12-24 | 2021-04-27 | 华中农业大学 | Porcine infectious actinobacillus pleuropneumoniae subunit vaccine |
CN113980101A (en) * | 2021-09-11 | 2022-01-28 | 江苏南农高科技股份有限公司 | Actinobacillus pleuropneumoniae subunit vaccine |
CN118388664A (en) * | 2024-05-21 | 2024-07-26 | 广东省农业科学院动物卫生研究所 | Haemophilus parasuis fusion antigen and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000012718A1 (en) * | 1998-08-27 | 2000-03-09 | Microbiological Research Authority | Superoxide dismutase as a vaccine antigen |
CN101691396A (en) * | 2009-09-16 | 2010-04-07 | 天津农学院 | Recombination outer membrane protein, coding gene and expression vector of porcine actinobacillus pleuropneumoniae (APP) and preparation method thereof |
-
2018
- 2018-06-13 CN CN201810608304.4A patent/CN108794584B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000012718A1 (en) * | 1998-08-27 | 2000-03-09 | Microbiological Research Authority | Superoxide dismutase as a vaccine antigen |
CN101691396A (en) * | 2009-09-16 | 2010-04-07 | 天津农学院 | Recombination outer membrane protein, coding gene and expression vector of porcine actinobacillus pleuropneumoniae (APP) and preparation method thereof |
Non-Patent Citations (2)
Title |
---|
ACCESSION NO.: NC_010278 REGION: 1515756..1517504: "Actinobacillus pleuropneumoniae serovar 3 str. JL03, complete genome", 《GENBANK DATABASE》 * |
TAKASHI INUI等: "Morphological Changes and Lysis Induced by b-Lactams Associated with the Characteristic Profiles of Affinities of Penicillin-Binding Proteins in Actinobacillus pleuropneumoniae", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112704732A (en) * | 2020-12-24 | 2021-04-27 | 华中农业大学 | Porcine infectious actinobacillus pleuropneumoniae subunit vaccine |
CN112704732B (en) * | 2020-12-24 | 2022-02-25 | 华中农业大学 | Porcine infectious actinobacillus pleuropneumoniae subunit vaccine |
CN113980101A (en) * | 2021-09-11 | 2022-01-28 | 江苏南农高科技股份有限公司 | Actinobacillus pleuropneumoniae subunit vaccine |
CN113980101B (en) * | 2021-09-11 | 2023-06-30 | 江苏南农高科技股份有限公司 | Actinobacillus pleuropneumoniae subunit vaccine |
CN118388664A (en) * | 2024-05-21 | 2024-07-26 | 广东省农业科学院动物卫生研究所 | Haemophilus parasuis fusion antigen and application thereof |
CN118388664B (en) * | 2024-05-21 | 2024-12-03 | 广东省农业科学院动物卫生研究所 | Haemophilus parasuis fusion antigen and application thereof |
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