CN101241137A - A gold standard rapid diagnostic reagent for porcine cysticercosis - Google Patents

Abstract

The invention relates to a rapid diagnostic reagent for detecting pig cysticercosis (Cysticercosis) and a preparation method of the rapid diagnostic reagent. The diagnostic reagent of the present invention is a colloidal gold detection test strip. The Cysticercosis suis antigen used in the present invention adopts the recombinant antigen of Taenia solium prepared by artificial cloning, and the anti-cysticercosis suis immunoglobulin used is obtained by immunizing animals with the recombinant antigen of Taenia solium prepared by artificial cloning.

Description

A gold standard rapid diagnostic reagent for porcine cysticercosis

technical field

The invention relates to a rapid diagnostic reagent for detecting pig cysticercosis (Cysticercosis) and a preparation method of the rapid diagnostic reagent. The diagnostic reagent of the present invention is a colloidal gold detection test strip.

Background technique

Cysticercosis is a food-borne zoonotic parasite caused by the larvae of Taenia solium, Cysticercosis cellulosae, parasitizing in humans or intermediate hosts such as pigs and wild boars. sick. The disease is widespread worldwide and is an important public health problem in most parts of China. In my country, the disease is listed as a zoonotic disease that must be checked for meat import and export, slaughtered animals, and the Chinese government's proposal to let people eat "safe meat". According to the national survey of important human parasitic diseases completed by the Ministry of Health in 2004, the national average infection rate of tapeworms was 0.28%, which was 52.47% higher than the 0.18% found in the national human parasite distribution survey completed in 1990. The number of people infected with tapeworm was 550,000, of which 96,008 were investigated for cysticercosis, and the positive rate was 0.58%. Cysticercosis parasitizes the muscles and various tissues and organs of the human body and is harmful to human health. It usually causes symptoms such as epileptic seizures, increased intracranial pressure, mental disorders, and blindness. If it is not treated in time or improperly treated, it will often cause disability or even life-threatening . In addition, cysticercosis also restricts the development of pig industry and meat processing industry.

So far, post-mortem examination or immunological methods are widely used in China to detect circulating antibodies in serum to realize the quarantine of porcine cysticercosis. Among them, the antigens used in serological testing are all derived from cystic fluid or parasite antigens. The outstanding problems are that the collection of parasites is limited by objective conditions (such as difficult access, shortage of sources, and limited quantity), and the preparation of purified antigens is tedious. And the specificity and sensitivity are reduced, and the problems of false positive and false negative are often prone to occur in serological tests. The use of genetically engineered recombinant antigens to prepare diagnostic reagents is an ideal way to solve the immunodiagnosis of cysticercosis. Cysticerc macromolecular proteins have low specificity due to cross-reactivity with other tapeworms in the diagnosis, so the current research mainly focuses on low molecular weight proteins (usually less than 30ku) cysticercosis diagnostic antigens, such as 8ku protein, see Kathy Hancock, Azra Khan , Fatima B.Williams, et al.Characterization of the 8-Kilodalton Antigens of Taenia solium Metacestodes and Evaluation of Their Use in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis[J].Journal of Clinical Microbiology.2003,41(6):2577- 2586, 10ku protein, see Chung JY, Bahk YY, Huh S, et al.A recombinant 10kDa protein of Taenia solium metacestodes specific to active neurocysticercosis[J].The Journal of Infectious disease, 2000, 181(5): 1870-1872, 14ku protein, see Marcia Ramos Monteiro da Silva, Augusto Mendes Maia, et al. Recombinant expression of Taenia solium TS14 antigen and its utilization for immunodiagnosis of neurocysticercosis [J]. Acta Tropica, 2006, 100 (3): 192-198, 24ku protein, see Kathy Hancock, Sowmya Pattabhi, Fatima W, al. Characterization and cloning of T24, a Taenia solium antigen diagnostic for cysticercosis [J]. Molecular and Biochemical Parasitology, 2006, 147(1): 109-117, etc.

Ante-mortem diagnosis of cysticercosis is very difficult. Existing immunological diagnostic methods such as indirect hemagglutination test, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunoblotting (ELIB) and other methods have limitations in operating procedures and adaptability. Certain deficiencies, such as ELISA detection has high requirements for detection equipment, environmental conditions, and operational skills, and is also limited by factors such as long detection cycle and high cost of small batch detection, so it is still difficult to popularize and apply it in pig slaughtering enterprises. Therefore, finding a simple, fast, accurate and field-operable detection method is an important technical guarantee for effectively implementing animal product safety supervision and ensuring the implementation of these laws and regulations.

Colloidal gold immunochromatography technology is a rapid development in the past ten years that combines colloidal gold labeling technology, immunoassay technology, chromatography analysis technology, monoclonal antibody technology and new material technology. A new type of in vitro diagnostic technology. It has the advantages of simple and fast, clear results, no need for complicated operation skills and special equipment, and is known as "condensed ELISA". It has become a new direction in the field of clinical and quarantine diagnosis. This technology is established on the basis of immune diafiltration technology. It just changes the original longitudinal diafiltration to horizontal chromatography, but it is this improvement that drives the revolution of qualitative diagnostic reagents.

Domestic reports on the application of colloidal gold technology in the detection of porcine cysticercosis mainly include: Zhang Honghua et al. Journal of Parasitic Disease Control. 1994, 7(1): 35-37.) Using gold-labeled porcine cysticercis cyst fluid antigens, using monoclonal antibodies to detect circulating antigens in the cerebrospinal fluid of patients with cerebral cysticercis, the positive rate can reach 88.09%. Liu Yubing (Liu Yubing, Xu Hongxiu, Wang Changyuan, etc. Application of gold standard immunofiltration method in the diagnosis of cerebral cysticercosis [J] Endemic Disease Bulletin. 2001, 16(2): 11-13. Liu Yubing, Zhang Honghua, Xu Hongxiu, etc. Study on detection of circulating antigens in patients with cysticercosis by assay[J]. Chinese Journal of Zoonoses. 2002, 18(2): 84-86.) Application of two monoclonal antibodies against porcine cyst fluid antigens , established a gold-standard immunofiltration method to detect circulating antigens in cerebrospinal fluid, and the positive rate reached 77.8-93.75%; later established a gold-standard immunochromatography method, and the positive rate for detecting active cerebral cysticercosis reached 80.77%. The seropositive rate of neurocysticercosis patients was 55%. Yu Ting et al. (Yu Ting, Liu Sufang, Huang Jing, et al. Research on the detection of Cysticercosis antibodies in serum by rapid dot immunogold filtration method [J]. Chinese Public Health. 2000, 16(12): 1139-1140.) Gold standard method and ELISA were compared to test 57 serum samples of patients with cysticercosis, 54 samples were positive by both methods, and 1 sample was negative by both methods, the total coincidence rate was 95%. Huang Bingcheng et al. (Huang Bingcheng, Li Guiping, Jia Fengju, et al. Research on the diagnosis and curative effect evaluation of cerebral cysticercosis by dipping test of gold-labeled anti-human monoclonal antibody [J]. Chinese Journal of Parasitic Disease Control. 2000, 13(4): 273-275 .) 80 cases of active neurocysticercosis patients were detected with gold-labeled anti-human IgG4 monoclonal antibody, and the positive rate was 91.3%; 19 cases of inactive patients were detected, and 2 cases were positive. Yuan Jianhua et al. (Yuan Jianhua, Gan Shaobo, Chen Zhiyong. Development and assessment of cysticercosis IgG colloidal gold diagnostic kit (chromatographic method) [J]. Chinese Journal of Zoonoses. 2001, 17(6): 59-61.) The IgG gold standard chromatography (one-step method) diagnostic kit successfully developed for the diagnosis of cysticercosis at home and abroad has a sensitivity of 93.23% and a specificity of 97.37% through clinical assessment. The kit detects hydatid serum The cross-reaction rate is 45%, and there is no cross-reaction with sera of parasitic patients such as schistosomiasis, liver fluke, and lung fluke. Tang Yude et al. (Tang Yude, Zhou Dongming, Lu Chengping. Establishment and application of gold immunochromatography method for rapid detection of cysticercosis antibody [J]. Chinese Journal of Zoonoses. 2003, 19(5): 77-79. ) established a gold immunochromatographic method for the rapid detection of Cysticercosis antibodies in pigs, and compared with ELISA, the coincidence rate of the two detecting Cysticercosis antibodies was 96.8%. The above methods all suggest that the immunocolloidal gold rapid diagnosis method is simple, rapid, suitable for grass-roots promotion and application, and can be used for the diagnosis and epidemiological investigation of cysticercosis.

Chinese invention patent application 200610050841.9 discloses a gold-labeled immunofiltration kit for diagnosing porcine cysticercosis spot and its preparation and application method. The patent application uses nitrocellulose membrane as a solid phase carrier to absorb pig blood samples to be tested. Using the combination of cysticercosis antigen and colloidal gold as the liquid phase, and as a probe and indicator at the same time, when the antigen is combined with the corresponding antibody ligand, gold particles will gather at the antibody ligand site in large quantities, forming a red color visible to the naked eye spot. In the application of the kit for this patent application, 1 μL of the dry paper extract of the tested pig blood sample or serum dilution is spotted on the nitrocellulose membrane, 100 μL of the washing solution is added, and 100 μL of colloidal gold of the porcine cysticercosis antigen is added dropwise for 2-3 minutes. The red spot is formed or not formed in the blood, the result is easy to judge, the detection cost is low, it has the advantages of sensitivity, specificity, high detection rate, labor saving, time saving, and convenient blood sampling, etc. It is suitable for the diagnosis and circulation of porcine cysticercosis Applications in market quarantine and other fields.

However, the diagnostic antigens used in the above reports are all cystic fluid antigens or insect body antigens. The prominent problems are that the source of antigens is difficult, the purification is cumbersome, and false positives or false negatives are prone to occur. However, the preparation of monoclonal antibodies against cystic fluid antigens of porcine cysts requires high production costs, and the preservation requirements of monoclonal antibody cell lines are relatively high, which is prone to loss.

Contents of the invention

The invention provides a colloidal gold detection test strip for cysticercosis suis and a preparation method thereof which can overcome the deficiencies of the prior art.

The test strip of the present invention comprises a sample-loading water-absorbing layer made of porous fiber material which is sequentially fixed end to end on the PVC liner material, a sample-loading water-absorbing layer located at the tail end of the sample-loading water-absorbing layer and connected with the sample-loading water-absorbing layer. The gold-labeled binding pad with the antigen, the reaction pad made of nitrocellulose membrane connected to the tail end of the gold-labeled binding pad, and the absorbent layer made of water-absorbing material connected to the tail end of the reaction pad, wherein the reaction pad is coated with A detection area composed of Cysticercosis antigen and a quality control area downstream of the detection area composed of Cysticercosis antibody. The Cysticercosis suis antigen used in the present invention adopts the recombinant antigen of Taenia solium prepared by artificial cloning, and the anti-cysticercosis suis immunoglobulin used is obtained by immunizing animals with the recombinant antigen of Taenia solium prepared by artificial cloning.

The pig cysticercosis antigen used in the cysticercosis gold standard test strip of the present invention can be artificially prepared TSOL18 recombinant antigen of Taenia solium. And this TSOL18 recombinant antigen of Taenia solium is that the 18ku coding gene derived from Taenia solium is subjected to RT-PCR by using genetic engineering technology, and the PCR product is constructed on a eukaryotic expression vector to obtain a recombinant plasmid. The recombinant plasmid was electrotransformed into yeast competent cells to obtain a recombinant strain, and then the recombinant bacteria were induced to express the recombinant protein, which was used to prepare the gold-labeled antigen and the coated detection area; the application of genetic engineering technology will be derived from Taenia suis. Perform PCR on 18ku gene, then clone the PCR product into an expression vector to construct a prokaryotic expression vector, obtain a fusion protein after induced expression, immunize animals with the fusion protein, extract the serum of the immunized animal, and isolate the anti-fusion protein antibody from it, use this polyclonal Antibody coated quality control area.

In the embodiment of the present invention, the porcine cysticercosis antigen used is to carry out RT-PCR on the 18ku gene derived from Taenia solium hexacarpa, and the obtained product is constructed into a eukaryotic expression vector pPIC9K-TSOL18, which is electrotransformed into Pichia pastoris GS115. State cells, the recombinant protein obtained by induced expression; and using genetic engineering technology to perform PCR on the 18ku gene derived from Taenia solium, and then clone the PCR product into the expression vector pGEX-4T-1 to construct the prokaryotic expression vector pGEX - TSOL18/BL21, the fusion protein is obtained after induced expression, the animal is immunized with the fusion protein, the serum of the immunized animal is extracted, and the anti-fusion protein antibody is isolated from it, and the quality control area is coated with the polyclonal antibody.

The Cysticercosis suis gold standard test strip of the present invention can also be provided with a whole blood filtering device on the sample adding water-absorbing layer.

The preparation method of cysticercosis suis gold standard test strip of the present invention is:

a. Collect the eggs of Taenia solium, hatch and activate them by chemical method, extract the total RNA of Hexacarpa, amplify the TSOL18 coding gene by RT-PCR, and design and synthesize expression primers according to the nucleic acid sequence of the TSOL18 gene;

b. recovering the PCR amplification product of step a; carrying out double digestion with the recovered PCR amplification product with a restriction endonuclease, constructing the PCR product into a eukaryotic expression vector to obtain a recombinant plasmid, and electrotransforming the recombinant plasmid into yeast Competent cells to obtain recombinant strains, and then induce the expression of recombinant bacteria to obtain recombinant antigen proteins;

c. Recover the PCR product obtained in step a, then clone the PCR product into an expression vector to construct a prokaryotic expression vector, induce expression to obtain a fusion protein, immunize animals with the fusion protein, extract the serum of the immunized animal, and isolate polyclonal antibodies against the fusion protein therefrom ;

d. Prepare colloidal gold, and then use the prepared colloidal gold and the recombinant antigen obtained by b to prepare a gold-labeled antigen;

e. Fully adsorbing the gold-labeled recombinant antigen obtained in d to a porous water-absorbing material to obtain a gold-labeled binding pad;

f. Set the detection area coated with the recombinant antigen obtained in b and the quality control area coated with the polyclonal antibody obtained in step c on the reaction pad of the nitrocellulose membrane respectively, and make the detection area on the reaction pad coincide with the quality control area according to the design requirements. There is a blank area between the control areas;

g. Fix the water-absorbing material, the gold standard bonding pad, the nitrocellulose membrane reaction pad and another section of water-absorbing material on the substrate of the impermeable material in sequence, so that the materials are kept connected end to end, and the detection area on the reaction pad is located at the adding point. On the sample side, the fixed substrate was cut into required strips according to the design requirements to obtain the described Cysticercosis suis gold standard test strip.

In the embodiment given by the present invention, the preparation method of the gold standard test paper strip of cysticercosis suis is:

a. Using the total RNA of Taenia suis as a template, RT-PCR was performed with upstream primer R: 5′TCT CTC CGAAAC AAT GAG TTA 3′ and downstream primer F: 5′TAA TAG ATA CCC ATT TTC ATCACA 3′ , the resulting product is purified and recovered;

b. Construct the cloning vector pGEX-TSOL18 with Taenia suis TSOL18 obtained in a;

c. Use the pGEX-TSOL18 plasmid as a template to

Upstream primer P1: 5'CGA GAATTC GACATTTTCGTTCCATACCTT3'

EcoRI

Downstream primer P2: 5'TAG CGGCCG CTCACTACGATCTTCGGACCTTC3'

NotI

Carry out PCR amplification, and the obtained product is purified and recovered;

d. Take the purified product obtained in step C and the pPIC9K vector plasmid respectively with EcoRI/NotI double enzyme digestion, and then use Agarose Gel DNA Extraction Kit to recover and ligate, and the ligated product is transferred into Escherichia coli JM109 competent cells to obtain the pPIC9K-TSOL18 plasmid, and then Linearized with Sal I single enzyme digestion, electrotransformed Pichia pastoris competent cells, and obtained recombinant multi-copy strain pPIC9K-TSOL18/GS115;

e. Continuously culture pPIC9K-TSOL18/GS115 in a fermenter to obtain TSOL18 recombinant protein;

f. Use the pGEX-TSOL18 obtained in step b as a template to amplify the TSOL18 coding gene, construct the prokaryotic expression vector pGEX-TSOL18/BL21, collect the bacteria after induction and expression, immunize rabbits after purification, collect and separate immune rabbit serum, and purify to obtain IgG of rabbit anti-TSOL18-GST polyclonal antibody;

g. prepare colloidal gold, and then use the prepared colloidal gold and the recombinant antigen obtained by e to prepare a gold-labeled antigen;

h. Fully adsorbing the gold-labeled recombinant antigen obtained in g into a porous water-absorbing material to obtain a gold-labeled binding pad;

i. On the reaction pad of the nitrocellulose membrane, respectively set the detection area coated with the recombinant antigen obtained in e and the quality control area coated with the polyclonal antibody obtained in step f, and make the detection area on the reaction pad and the quality control area according to the design requirements There is a blank space between the zones;

j. Fix the water-absorbing material, gold standard binding pad, nitrocellulose membrane reaction pad and another section of water-absorbing material on the substrate of the impermeable material in sequence, so that the materials are connected end to end, and the detection area on the reaction pad is located On the sample side, the fixed substrate was cut into required strips according to the design requirements to obtain the described Cysticercosis suis gold standard test strip.

In the preparation method of the Cysticercosis suis gold standard test strip of the present invention, a whole blood filtering device can also be arranged on the sample-applying water-absorbing layer.

Compared with the prior art, the present invention has the following advantages:

1. The present invention adopts artificial cloning to prepare recombinant cysticercosis antigen, which solves the problems of difficult source of antigen in the prior art, complicated purification, and prone to false positives or false negatives. The preparation of cysticercosis antigen by artificial cloning is not only easy to source, low in cost, but also It can be fermented and produced in batches, and the quality is easy to control. In particular, the detection box for the preparation and detection of the recombinant antigen of Taenia solium sulum TSOL18 utilized in the present invention not only has the advantages of single component, strong specificity, low cost, and can be prepared in large quantities in the laboratory, but also can conveniently resolve the antigen source problem.

2 In a specific example of the present invention, the eukaryotic expression vector pPIC9K-TSOL18/GS115 was constructed, the glycosylation modification of the expressed protein was realized, and the recombinant protein TSOL18 with natural protein characteristics was obtained, and it had good immune activity. It can be highly expressed at the fermenter level, and the amount of TSOL18 recombinant protein can be obtained per liter of fermentation broth is 2.54g/L; after purification by SephacrylS-300HR, a single band with a size of about 16ku can be obtained, and the purity of TLC scanning analysis exceeds 90%. The TSOL18, which has natural protein properties and can be mass-produced, has a good application prospect and popularization value in the preparation of test paper for detecting porcine cysticercosis or other detection devices.

3. The recombinant antigen prepared by the present invention for immunizing animals has a larger molecular weight than TSOL18, so it has better immunogenicity, and it is easy to obtain high-titer rabbit anti-TSOL18-GST polyclonal antibody after immunizing rabbits.

4 There is no domestic report on the application of TSOL18 as a diagnostic antigen to detect porcine cysticercosis. The reported recombinant proteins are all from the cysticercosis stage of Taenia solium, and the E. coli expression system cannot be glycosylated, which affects the antigenic activity of the recombinant protein, and the carrier part of the fusion protein is prone to cross-reaction, resulting in false positives. The disadvantages of the prior art can be overcome by selecting a eukaryotic expression system.

5. The gold standard test strip of the present invention adopts a one-step method when detecting porcine cysticercosis, has higher specificity and sensitivity, and its detection is fast, and the result judgment is completed within 10-15min, without the need for special experimental facilities, experimental conditions and dedicated operators.

6. The test strip preparation method of the present invention has the characteristics of easy manufacture, good stability and repeatability. In particular, the test strip of the present invention is provided with a whole blood filtering device, which can greatly facilitate on-site use.

Description of drawings

Fig. 1 is process roadmap of the present invention

Fig. 2 is a structural schematic diagram of the present invention, wherein: 1 is the water-absorbing filter paper layer of the handheld terminal, 2 is the NC membrane reaction cushion, 3 is the gold standard recombinant antigen cushion, 4 is the glass fiber water-absorbing layer of the testing end, and 5 is the quality control line , 6 is a detection line, and 7 is a PVC liner.

Fig. 3 is the result judgment schematic diagram of the present invention, wherein: + represents the porcine standard positive serum reaction result; ± represents the suspicious porcine serum sample reaction result; - represents the porcine standard negative serum reaction result; In this case, the test strip is invalid and cannot be used for detection.

Fig. 4 is a construction diagram of the eukaryotic expression vector pPIC9K-TSOL18/GS115.

Figure 5 is a PCR amplification map of the TSOL18 gene, in which: 1 is the DL2000 nucleic acid marker; 2 is the TSOL18 gene obtained by RT-PCR.

Figure 6 shows the enzyme digestion identification and PCR analysis of the recombinant expression plasmid pPIC9K-TSOL18, in which: 1 is the PCR product of TSOL18; 2 is the PCR product of EcoRI and NotI double digestion pPIC9K-TSOL18; 3 is the DL2000 nucleic acid marker.

Fig. 7 is the PCR identification of the recombinant strain, wherein: 1 is the PCR product of the pPIC9K-TSOL18/GS115 recombinant strain; 2 is the DL2000 nucleic acid marker.

Figure 8 is the SDS-PAGE electrophoresis of TSOL18 expression protein and purification, in the figure: M is a low molecular weight protein standard; 1 is the expression product TSOL18 induced by methanol in a 5L fermenter for 72 hours; 2 is SephacrylS-300HR column chromatography after purification TSOL18 protein.

Figure 9 is the expression product of TSOL18-GST and its purified SDS-PAGE electrophoresis diagram, in which: M is the protein molecular weight standard; 1 is the affinity purified TSOL18-GST fusion protein; 2 is the lysed supernatant after induction; 3 is After induction, the cells were lysed and precipitated.

Figure 10 is the SDS-PAGE electrophoresis image of purified rabbit anti-TSOL18-GST IgG, wherein: M is a low molecular weight protein standard; 1 is the IgG of purified rabbit anti-TSOL18-GST protein.

Detailed ways

The present invention is described in further detail in conjunction with embodiment:

Example 1 (Preparation Example of Recombinant Diagnostic Antigen)

Cloning of TSOL18 Gene of Taenia solium and Construction of Eukaryotic Expression Vector pPIC9K-TSOL18/GS115

1 Cloning of the TSOL18 gene of Taenia solium

1.1 Design and synthesis of PCR primers

According to the nucleotide sequence of Taenia suis TSOL18 (AF017788) registered on GeneBank, a pair of primers R and F were designed with DNAStar software. The upstream primer R starts from the start codon of the coding region, and the downstream primer F is the sequence of the 3' non-coding region. The amplified fragment between the upstream and downstream primers includes the complete reading frame of the TSOL18 gene. Primers were synthesized by Bao Biological Engineering (Dalian) Co., Ltd.

Upstream primer R: 5′TCT CTC CGA AAC AAT GAG TTA 3′

Downstream primer F: 5′TAA TAG ATA CCC ATT TTC ATC ACA 3′

1.2 Amplification and recovery of TSOL18 coding gene of Taenia solium

The eggs of Taenia solium were collected, hatched and activated by chemical method, the total RNA of Hexacarcinus was extracted by Trizol method, and the TSOL18 gene was amplified by RT-PCR reverse transcription.

The RT-PCR amplification system was: 10 μl of Total RNA suspension, 1 μl of OligodT primer, pre-denatured at 85°C for 5 minutes after mixing, and quickly placed in an ice bath for 5 minutes. Then add 4 μl of 5× reverse transcriptase buffer, 0.5 μl of RNasin (50u/μl), 1 μl of 2.5mM dNTP Mixture, 1 μl of reverse transcriptase (AMV) (10u/ul), sterilized DEPC water to 20 μl, and mix After homogenization, react in a water bath at 42°C for 1 hour, and then inactivate the reverse transcriptase at 95°C for 5 minutes.

The double-stranded cDNA PCR amplification system is: reverse transcription product 10μl, 10×PCR buffer (Mg ²⁺ free) 5μl, 2.5mM dNTP Mixture 2μl, upstream and downstream primers (50pmol/μl) each 1μl, 25mM MgCl ₂ 3μl, inactivated Bacterial double distilled water was added to 50 μl. The PCR reaction conditions were as follows: after pre-denaturation at 95°C for 5 min, 1 μl (5 U/μl) Taq DNA polymerase was added, followed by 30 cycles (94°C for 1 min, 55°C for 30 s, 72°C for 5 min), and finally 72°C for 5 min. After the amplification was completed, 5 μl of the PCR product was taken for electrophoresis analysis on a 1.2% agarose gel (containing 0.5 μg/ml ethidium bromide, EB), and a TSOL18 gene fragment (Fig. 5) with a size of about 393 bp was amplified; Purified and recovered by DNA Gel Rapid Recovery Kit.

2 Construction of the cloning vector pGEX-TSOL18 of Taenia solium Hexacarpa TSOL18

Take 1 μl (5 μg/μl) of the pGEM-T easy cloning vector (purchased from Promega) and mix it with 3 μl of the target fragment recovered and purified from the gel, add 5 μl of 2×rapid ligase buffer, 1 μl of _T4 DNA ligase (3u/μl), and mix After homogenization, place at 4°C overnight for connection; transform Escherichia coli JM109, screen positive clones, extract a small amount of plasmid by alkaline lysis, and confirm its size after agarose gel electrophoresis, PCR amplification and restriction endonuclease digestion identification As expected, the sequence of Shanghai Jingtai Biotechnology Company confirmed that the fragment has no base mismatch. The sequence was analyzed by DNAstar software, and compared with the published sequence, it was confirmed that the homology was 99.45%.

3 Construction of the eukaryotic expression vector pPIC9K-TSOL18/GS115 of Taenia solium hexacarpa TSOL18

3.1 Design and synthesis of expression primers

According to the nucleic acid sequence of the sequenced TSOL18 gene, use DNA Star software to design expression primers outside its reading frame, and remove the 51bp signal peptide sequence at the 5' end (this will not have much impact on the structure and function of the encoded protein can also increase soluble expression to a certain extent), and according to the codon preference of Pichia pastoris (see Zhao Xiang, Hawke Ke, Li Yuyang. Analysis of the code usage of Pichia pastoris. Acta Bioengineering, 2000, 16( 3): 308-311), under the premise of not changing its amino acid sequence, low-utilization codons GAT and GTG were synonymously mutated into high-utilization codons GAC and GTT, and EcoRI and NotI were added to the 5' end of the expression primers respectively Restriction sites and 6 protective bases. The amplified fragment between the upstream and downstream primers is 342bp. Primers were synthesized by Treasure Biological (Dalian) Engineering Company.

TSOL18 gene

Upstream primer P1: 5'C GAGAATTC GACATTTTCGTTCCATACCTT3'

EcoRI

Downstream primer P2: 5'TAG CGGCCG CTCACTACGATCTTCGGACCTTC3'

NotI

Primers for pPIC9K plasmid vector:

5′AOX 1,5′-GACTGGTTCCAATTGACAAGC-3′

3′AOX 1,5′-GCAAATGGCATTCTGACATCC-3′

The pPIC9K plasmid vector primers were used to carry out PCR amplification using the recombinant multi-copy strain genomic DNA as a template to prove whether the TSOL18 gene was integrated into the GS115 chromosomal DNA.

3.2 Construction of expression vector pPIC9K-TSOL18/GS115

Using the correctly sequenced pGEX-TSOL18 plasmid as a template, the TSOL18 fragment was amplified with primers P1 and P2 with restriction sites. 50 μL of PCR amplification system, including 1 μL of template, 5 μL of 10×PCR buffer, 0.5 μL of P1 and P2 primers (20 μM), 1 ul of Taq enzyme, 4 μL of 2.5mM dNTP, 4 μL of 25mM MgCl ₂ , and sterilized double-distilled water to 50 μL. Amplification conditions: pre-denaturation at 95°C for 5 min; 35 cycles (94°C for 50 s; 56°C for 40 s; 72°C for 1 min); extension at 72°C for 10 min. After the amplification is completed, take 5 μL of the PCR product and detect it by electrophoresis on a 1.0% agarose gel (containing 0.5 μg/mL ethidium bromide, EB) to obtain a specific DNA fragment of 342 bp; Purification and recovery.

The PCR purified product and the pPIC9K vector plasmid were digested with EcoRI/NotI respectively, recovered with AgaroseGel DNA Extraction Kit and ligated, and the ligated product was transformed into Escherichia coli JM109 competent cells (prepared by CaCl ₂ method). Ligation system: 7 μL of digested target fragment, 1 μL of enzyme-digested vector, 1 μL of 10×Ligationbuffer, 1 μL of T ₄ DNA (3u/μL) ligase, mix well and place at 16°C for 12 hours, transform Escherichia coli JM109, screen positive clones, alkali A small amount of plasmid was extracted by lysis method. After the recombinant plasmid pPIC9K-TSOL18 was amplified by PCR and identified by restriction endonuclease EcoR I/Xho I double digestion, a specific band of 342bp appeared, which was consistent with the size of the expected target fragment (Figure 6). The recombinant strains identified as positive by enzyme digestion were sent to Dalian Bao Biological Engineering Co., Ltd. for sequencing, and the results were in full agreement with expectations. Linearize pPIC9K-TSOL18 with Sal I single enzyme digestion, electrotransform Pichia pastoris competent cells, the colonies grown on MD medium are His ⁺ transformants, and gradually screen for G418 resistant strains with high copies of the target gene by photocopying recombinant strains.

Using the recombinant multi-copy strain genomic DNA as a template, perform PCR amplification on the TSOL18 gene integrated into the chromosomal DNA of Pichia pastoris GS115, PCR amplification system: 10×buffer 2.5 μL, 5′AOX1 primer (50 pmol/μL) 0.5 μL , 3′AOX1 primer (50pmol/μL) 0.5μL, genomic DNA template 2.5μL, Taq enzyme 0.5μL, dNTP 2μL, sterilized water to make up the volume to 25μL; amplification conditions: 95°C pre-denaturation, 5min; 35 cycles ( 94°C for 1min, 55°C for 1min, 72°C for 1min); then extend for 10min at 72°C. After the amplification, 5 μL of the reaction solution was electrophoresed on agarose gel to obtain a specific fragment of 834 bp (the blank strain should be 492 bp), indicating that the TSOL18 gene was indeed integrated in the transformant genome (Figure 7), and the expression vector pPIC9K-TSOL18/GS115 The construction process is shown in Figure 4.

4 Induced expression and purification of TSOL18 recombinant protein from Taenia solium

4.1 Induced expression of TSOL18 recombinant protein

Recombinant yeast pPIC9K-TSOL18/GS115 was continuously cultured in a 5L fermenter with a working volume of 3L. The culture process was as follows:

Pick the single clone of the seed culture and inoculate it in 20mL of YPD medium, culture it on a shaker at 28°C for 24 hours, then inoculate it in 200mL of YPD medium and continue to cultivate it for about ₁₈ hours. Inoculate in a 5L fermenter. During the cultivation process, the content of each substance is controlled by computer, the oxygen content is not less than 50%, and the pH value is 5.0. The concentration of methanol is controlled throughout the fermentation process: the methanol flow rate is 3mL/L/h for the first 2-4h of induction, the methanol flow rate for the first 4-8h of induction is 6mL/L/h, and the methanol flow rate is 8h after induction 9mL/L/h, a total of about 72h induction. After the end of the induction, centrifuge at 10000r/min for 30min to take the supernatant and analyze it by SDS-PAGE. The results show that the supernatant of the bacterial solution contains an expression product with a size of about 16ku ( FIG. 8 ). Concentrate and freeze-dry the bacterial supernatant, mark the batch number, and store at 4°C for future use. Each batch of fermentation can obtain recombinant protein 2.54g/L. In this way, the soluble and high-efficiency expression of the recombinant protein is realized, which lays the foundation for the subsequent protein purification process.

4.2 Purification of TSOL18 recombinant protein

After loading the pretreated Sephacryl S-300 (polyacrylamide-sephadex S-300) into the column, it was fully balanced with 0.01MPBS (pH7.2) buffer until the conductivity, UV absorption baseline and pH were all stable. Take 0.5 mL of the induced expression product of the recombinant strain (about 25 mg protein content), elute with the same buffer, flow rate is 0.7 mL/min, pressure is 0.2 Mpa, and peak samples are collected. The protein content was determined using a UV spectrophotometer, and the correction formula was as follows:

Protein content (mg/mL) = (1.45×OD280-0.74×OD260)×dilution factor

The purity of the protein was analyzed by SDS-PAGE, and the results showed that the purified target protein presented a single band (Figure 8), and the protein recovery was relatively large, and about 0.854 g of purified protein could be obtained per 1000 mL of culture supernatant.

Embodiment 2 (preparation and purification embodiment of rabbit anti-TSOL18-GST IgG)

1. Preparation of TSOL18-GST fusion protein

The cloning of the gene encoding the target protein TSOL18 and the construction of the cloning vector pGEX-TSOL18 are the same as in Example 1, and the expression vector primers used are designed as follows:

P3: 5'ccggaattcagcggtgaccgtacattcgg3 '

EcoRI

P4: 5'ccg ctccga gctacgaacggcggaccttcttgt3'

XhoI

The TSOL18 target gene was cloned into the expression vector pPGEX-4T-1, and the prokaryotic expression vector pPGEX-TSOL 18/BL21 was constructed. After being induced and expressed by 0.1mmol/L IPTG at 28°C for 6h, the bacterial liquid was collected; the supernatant was sonicated and then purified by a GST-FF affinity column to obtain a high-purity TSOL18-GST fusion protein with a molecular weight of about 38ku (Figure 9 ); compared with TSOL18, this protein has a larger molecular weight and better immunogenicity. After immunizing rabbits, it is easy to obtain a high-titer rabbit anti-TSOL18-GST polyclonal antibody, which will be used for the preparation of quality control lines.

2 Preparation of rabbit polyclonal antibody against TSOL18-GST fusion protein

Choose 10 healthy young female qingzilan rabbits, weighing about 3kg/only. The same batch of purified TSOL18-GST recombinant antigen was combined with Freund's complete adjuvant and Freund's incomplete adjuvant according to the method of doubling the antigen dose to make a vaccine for routine immunization of rabbits. The immunization interval was 3 weeks, and at the end 10 days after the first immunization, the agar double diffusion test was carried out. When the titer reached 1:32-1:64, the blood was collected from the heart and the serum was separated.

3 Purification of rabbit anti-TSOL18-GST fusion protein IgG

The hyperimmune serum IgG was purified by a combination of saturated ammonium sulfate precipitation and Sephacryl S-300HR gel chromatography. The specific operation is as follows:

10 mL of hyperimmune serum was taken, and after preliminary purification by successive precipitation with 50% and 33% saturated ammonium sulfate, rabbit hyperimmune serum IgG was further purified by Sephacryl S-300HR gel chromatography. In SDS-PAGE analysis, there is only one band at the molecular mass slightly less than 66ku, which should be γ globulin, that is, the band of purified IgG. The purity of the IgG was more than 90% by thin-layer scanning, indicating that the purity of the IgG met the requirements for the preparation of test strips (Figure 10). This antibody is used for quality control lines coated on nitrocellulose membranes.

Embodiment three (test strip/card assembly preparation example)

Assembling and preparation method of gold standard rapid diagnostic reagent for porcine cysticercosis

1 Assembly of reagents

With reference to Fig. 2, the assembling of porcine cysticercosis gold label rapid diagnostic reagent is to paste the filter paper of hand-held end water-absorbing layer (1) and the glass fiber of test end water-absorbing layer (4) respectively at the two ends of PVC liner (7), and Paste a nitrocellulose membrane in the middle section to make an NC membrane reaction cushion (2), and sandwich a gold-labeled recombinant antigen cushion (3) between the test end glass fiber water-absorbing layer (4) and the NC membrane reaction cushion (2). ) glass fiber, 4/5 of which is clamped on the water-absorbing layer (4) of the test end, and 1/5 is pressed on the NC membrane of the reaction pad (2), and then the water-absorbing layer (1) at the hand-held end reacts with the NC membrane Attach the self-adhesive colored handle paper between the cushion layers (2), and attach the self-adhesive MAX line adhesive strip between the test end glass fiber water-absorbing layer (4) and the NC membrane reaction cushion layer (2), Cut into strips with a width of 3mm by a chopping machine, or make into cassettes, and store in a drying cylinder at 4°C.

The preparation of the described gold-labeled recombinant antigen cushion layer (3) comprises: i preparation of colloidal gold, adding 0.6% of its volume, concentration of 1% tricitric acid in the concentration of 0.01%-0.03% Sodium, boiled for 5-10min, reduced to 20-40nm colloidal gold atomic solution, adjusted to pH 8.0-8.2; ii For colloidal gold labeling of TSOL18 recombinant antigen, add 1.0mg TSOL18 recombinant antigen to 100mL colloidal gold solution, and then add Add PEG20000 solution with a final concentration of 0.05% to the colloidal gold solution, centrifuge at 2000rpm for 10min to remove the precipitate, take the supernatant and centrifuge at 10000rpm for 1h to obtain the precipitate, dissolve the precipitate in 0.02M, pH7.4 Tris-HCl at 8mL/100mL Solution to obtain antigen colloidal gold solution: which contains 0.25% bovine serum albumin, 0.02% sodium azide; iii. The gold-labeled TSOL18 recombinant antigen is evenly immersed in glass fiber with a film dispenser, and then dried at 37°C to obtain the gold-labeled recombinant antigen. Cushion (3).

The preparation of the NC membrane reaction cushion layer (2) is to spray the TSOL18 recombinant protein and rabbit anti-TSOL18-GST IgG with a concentration of 2mg/mL on the nitrocellulose membrane to form a detection area (6) and a quality control area with a film spotting machine. (5), the distance between the detection area and the quality control area is 5 mm; then block with 0.01M pH7.0 PBS solution containing 10% calf serum for 30 min, rinse and dry to obtain the NC membrane reaction cushion (2).

The test strip prepared above can also be provided with a whole blood filtration device on the sample-applying water-absorbing layer, and its specific method can be referred to.

The using method of test strip of the present invention is as follows:

1 Preparation of serum samples

Blood was collected from the jugular vein of pigs, incubated at 37°C for 30 minutes, and then placed at 4°C for 2 hours to fully analyze the serum. The pig serum to be tested was centrifuged at 3000rpm for 15min to avoid residual blood cells and hemolysis as much as possible. When used for pig slaughter inspection, blood can be collected directly at slaughter and serum can be separated. If there is a whole blood filtration device on the test strip used, whole blood can be tested directly.

2 Operation method

Take out the test strip (card) and place it flat on the table; take 50 μL of the serum to be tested and make a 2-fold dilution with the sample diluent (0.02M PB, pH7.4), and slowly add the sample to the colloidal gold test strip with a dropper On the hole, 10-15min to observe the result.

3 result judgment

As shown in Figure 3, the test area and the quality control area have red bands, which is a positive result; the test area has no band, and the quality control area has a red band, and the quality control area has a red band. is an invalid result.

Embodiment four (detection of pig serum sample)

The detection method of the test strip in this embodiment is carried out according to the operation steps described in the third embodiment, wherein the specificity test, the sensitivity test and the sensitivity test all use the ELISA detection method of Pancystis as a control to evaluate various indicators. The test results are shown in Table 1, Table 2 and Table 3.

The content of the worm attack test: select healthy 3-month-old Landrace piglets, and each pig is orally infected with 20,000 mature eggs of Taenia solium at one time, and gradually develops into mature cysticerci (cysticercus) after 90 days. Dissect the worms at different times after infection, count the number of worms in each pig, collect blood at the same time, separate the serum, and use a test strip to detect its negative and positive. Among them, 3 pigs were necropsied 1.5 years after worm infection; 7 pigs were necropsied 90 days after worm infection; 6 pigs were necropsied 70 days after worm infection. After necropsy in all infected pigs, different numbers of viable worms were detected, and some of the worms of pigs had calcified.

The post-mortem necropsy method and ELISA detection method for pancysticercosis were used as controls to evaluate the coincidence rate of pig serum in the challenge test. The test results are shown in Table 4.

Table 1 Specificity and sensitivity test results of test strips

serum sample Quantity (parts) Positive rate Test strips ELISA Positive serum for Cysticercosis porcine Toxoplasma gondii positive serum Porcine cysticercosis positive serum Porcine negative serum   2021220 15/200/24/123/20 19/200/22/121/20

Table 2 The sensitivity results of test strips and ELISA detection

  Serum dilution 1:1 1:2 1:4 1:8 1:16 1:32 1:64 Negative control (1:2) Positive ELISA Untested + + + + + - test strip + + + + + - - - Remark Positive sample OD450: 1:2, 1.012±0.327; 1:4, 0.893±0.326; 1:8, 0.709±0.349; 1:16, 0.555±0.362; 1:32, 0.409±0.291; 1:64, 0.262± 0.121; negative control: 0.168

The stability test result of table 3 test strip

Detection time (d) 1 3 5 7 11 13 15 30 60 90 120 150   4°C Positive samples (15) Negative samples (10) 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10-   Room temperature Positive samples (15) Negative samples (10) 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 5+10- // //   37°C Positive samples (15) Negative samples (10) 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- 15+10- // Remark When stored at 4°C for 180 days, the detection line and quality control line are clear, the gold release is complete, and the chromatographic speed is fast; when stored at room temperature for 60 days, the gold release rate begins to slow down, but the chromatographic speed is good; when stored at 37°C for 120 days , the quality control line is fuzzy, the gold release is slow, and the chromatography speed is good.

Table 4 test strips detect the results of challenge test pig serum

serial number 0541 0542 0543 0771 0772 0773 0774 0775 0776 0779 Attack time (month) 18 twenty four 30 3 3 3 3 3 3 3 The number of insects checked (pieces) 2587 2032 1685 1405 0 11 732 30 763 873 ELISA (OD ₄₉₂ ) 1.100 1.264 0.946 1.050 0.855 1.095 1.182 1.308 1.049 1.737 Test strips + + + ± - + + + + + serial number 0726 0727 0728 0732 0762 0763 Standard Negative Control 1 Standard Negative Control 2 Attack time (month) 2.5 2.5 2.5 2.5 2.5 2.5 / / The number of insects checked (pieces) 805 339 47 4 25 290 0 0 ELISA (OD ₄₉₂ ) 1.103 0.653 1.073 0.507 0.906 0.842 0.224 0.220 Test strips ± + + + + - -

Table 1 specificity and sensitivity test result, this test strip and ELISA measure the positive coincidence rate of 54 parts of pig sera to be 78.9% (15/19), the negative coincidence rate is 89.5% (17/19), the total coincidence rate It was 85.0% (17/20). But all of them crossed with porcine cysticercosis (there is no standard method to solve this problem in China), and the cross-reaction rate was 33.3% (4/12). It was confirmed that the detection specificity of the test strip was close to that of the ELISA method.

Table 2 sensitivity test shows that the test strip detects the maximum dilution of Cysticercosis suis standard positive serum is 1: 16, and its sensitivity is 1-2 titers lower than the ELISA sensitivity when detecting strong positive serum, showing that the test strip It has a sensitivity close to that of ELISA.

The test strip stability test in Table 3 shows that at 37°C, the test strip can be stored stably for 120 days; the best storage condition for the test strip is 4°C, which can significantly prolong the validity period. In addition, the same batch of randomly selected test strips was tested repeatedly for 3 times, and the test results were consistent, indicating that the method has good stability and little variation.

The results in Table 4 show that the positive coincidence rate of the test strip and the post-mortem insect counting method is 80.0% (12/15); the positive coincidence rate with the ELISA kit is 75.0% (12/16), which does not include the test strip. The paper strip judged as suspicious 2 samples.

From the measurement results of the above indicators, it can be seen that the rapid diagnostic test strip for Cysticercosis porcine has good specificity, sensitivity, repeatability and stability, and the detection effect is similar to that of the ELISA diagnostic kit for Pancysticercosis. , safe, suitable for rapid diagnosis of porcine cysticercosis, especially for rapid quarantine of pig slaughter.

Claims (8)

1, pork measles gold test strip bar, comprise that tandem array is fixed in the application of sample water accepting layer with the porous fibrous material formation on the PVC lining material successively, the gold-marking binding pad that is adsorbed with pork measles gold mark antigen that is positioned at application of sample water accepting layer tail end and links to each other with the application of sample water accepting layer, the reacting pad that is made of nitrocellulose membrane and the absorption layer that is made of absorbent material that links to each other with the reacting pad tail end link to each other with the gold-marking binding pad tail end, wherein on reacting pad, be coated with the detection zone that constitutes by cysticercosis cellulosae antigen respectively and be positioned at the Quality Control district that constitutes by pork measles antibody in detection zone downstream, it is characterized in that the taeniasis suis recombinant antigen of the used cysticercosis cellulosae antigen of the present invention for artificial clone's preparation, used anti-pork measles immunoglobulin (Ig) is to use the taeniasis suis recombinant antigen immune animal of artificial clone's preparation to obtain.

2, pork measles gold test strip bar according to claim 1 is characterized in that employed cysticercosis cellulosae antigen is the TSOL18 recombinant antigen of the taeniasis suis hexacanth embryo of artificial preparation.

3, pork measles gold test strip bar according to claim 2 is characterized in that:

A. employed cysticercosis cellulosae antigen is: the 18ku gene that adopts technique for gene engineering will come from the taeniasis suis hexacanth embryo carries out RT-PCR, the PCR product is building up to obtains recombinant plasmid on the carrier for expression of eukaryon, again the recombinant plasmid electricity is transformed into the yeast competent cell, obtain recombinant bacterial strain, the bacterium abduction delivering of will recombinating again obtains recombinant protein, with this protein Preparation gold mark antigen and the detected district of bag;

B. used antibody is: the 18ku gene that will come from the taeniasis suis hexacanth embryo with technique for gene engineering carries out RT-PCR, the PCR product cloning is arrived the expression vector establishment prokaryotic expression carrier, again prokaryotic expression carrier is carried out abduction delivering, obtain fusion, use the fusion protein immunization animal, extract the serum of immune animal, therefrom separate obtaining anti-fusion antibody, with this antibody sandwich Quality Control district.

4, pork measles gold test strip bar according to claim 3 is characterized in that:

A. used cysticercosis cellulosae antigen is that the 18ku gene that will come from the taeniasis suis hexacanth embryo carries out RT-PCR, makes up carrier for expression of eukaryon pPIC9K-TSOL18, and the products therefrom electricity transforms Pichia pastoris GS115 competent cell, the recombinant protein that obtains through abduction delivering;

B. used antibody is: the 18ku gene that will come from the taeniasis suis hexacanth embryo with technique for gene engineering carries out PCR, again the PCR product cloning is arrived expression vector pGEX-4T-1, make up prokaryotic expression carrier pGEX-TSOL18/BL21, obtain fusion through abduction delivering, fusion protein immunization animal with purifying, extract the serum of immune animal, therefrom separate obtaining anti-fusion antibody, with this antibody sandwich Quality Control district.

5, according to the described arbitrary pork measles gold test strip bar of claim 1 to 4, it is characterized in that being provided with the whole blood filtration unit at the application of sample water accepting layer.

6, the described pork measles gold test strip of claim 3 bar preparation method is characterized in that:

A. collect the taeniasis suis worm's ovum, after chemical method hatching and activating, extract the total RNA of hexacanth embryo,, and design and synthesize the expression primer according to the nucleotide sequence of TSOL18 gene through RT-PCR amplification TSOL18 encoding gene;

B. the pcr amplification product of recycling step a; After the pcr amplification product that reclaims carried out double digestion with restriction enzyme, the PCR product is building up to obtains recombinant plasmid on the carrier for expression of eukaryon, the recombinant plasmid electricity is transformed into the yeast competent cell, obtains recombinant bacterial strain, the bacterium abduction delivering of will recombinating again obtains recombinant antigen;

C. reclaim a step gained PCR product, the PCR product cloning to the expression vector establishment prokaryotic expression carrier, is obtained fusion behind abduction delivering, use the fusion protein immunization animal, extract the serum of immune animal, therefrom separate obtaining anti-fusion antibody;

D. prepare collaurum, prepare gold mark antigen with prepared collaurum with by b gained recombinant antigen;

E. the golden indicated weight group antigen with the d gained fully is adsorbed in the porous water-absorbing material, obtains gold-marking binding pad;

F. on the reacting pad of nitrocellulose filter, set respectively by the detection zone of b gained recombinant antigen bag quilt with by the Quality Control district of c step gained antibody sandwich, and make the zone of leaving one section blank between detection zone on the reacting pad and the Quality Control district by designing requirement;

G. absorbent material, gold-marking binding pad, nitrocellulose filter reacting pad and another section absorbent material are fixed on the substrate of impermeable material successively, make each storeroom keep tandem array, and make the detection zone on the reacting pad be positioned at the application of sample side, by designing requirement the substrate that fixes is cut into required bar, obtains described pork measles gold test strip bar.

7, pork measles gold test strip bar preparation method according to claim 4 is characterized in that:

A. the total RNA with the taeniasis suis hexacanth embryo is a template, carries out RT-PCR with upstream primer R:5 ' TCT CTC CGAAAC AAT GAG TTA 3 ' and downstream primer F:5 ' TAA TAG ATA CCC ATT TTC ATCACA 3 ', and the products therefrom purifying reclaims;

B. make up cloning vector pGEX-TSOL18 with a gained taeniasis suis hexacanth embryo TSOL18;

C. be template with the pGEX-TSOL18 plasmid, with

Upstream primer P1:5 ' CGA GAATTCGACATTTTCGTTCCATACCTT3 '

EcoRI

Downstream primer P2:5 ' TA GCGGCCGCTCACTACGATCTTCGGACCTTC3 '

NotI

Pcr amplification, the products therefrom purifying reclaims;

D. get C step gained purified product and pPIC9K vector plasmid and use the EcoRI/NotI double digestion respectively, reclaiming the back with Agarose Gel DNA Extraction Kit again connects, connect product and change escherichia coli jm109 competent cell over to, obtain the pPIC9K-TSOL18 plasmid, use the linearization of Sal I single endonuclease digestion again, electricity transforms the Pichia pastoris competent cell, and multi-copy strains pPIC9K-TSOL18/GS115 obtains recombinating;

E. pPIC9K-TSOL18/GS115 is carried out Continuous Cultivation in fermentation tank, obtain the TSOL18 recombinant protein;

F. be template with b step gained pGEX-TSOL18, amplification TSOL18 encoding gene makes up prokaryotic expression carrier pGEX-TSOL18/BL21, behind abduction delivering, collect thalline, immunize rabbit behind the purifying is gathered and the separating immune rabbit anteserum, and purifying obtains the IgG of the anti-TSOL18-GST polyclonal antibody of rabbit;

G. prepare collaurum, prepare gold mark antigen with prepared collaurum with by e gained recombinant antigen again;

H. the golden indicated weight group antigen with the g gained fully is adsorbed in the porous water-absorbing material, obtains gold-marking binding pad;

I. on the reacting pad of nitrocellulose filter, be provided with respectively by the detection zone of e gained recombinant antigen bag quilt with by the Quality Control district of f step gained polyclonal antibody bag quilt, and make the zone of leaving one section blank between detection zone on the reacting pad and the Quality Control district by designing requirement;

J. absorbent material, gold-marking binding pad, nitrocellulose filter reacting pad and another section absorbent material are fixed on the substrate of impermeable material successively, make each storeroom keep tandem array, and make the detection zone on the reacting pad be positioned at the application of sample side, by designing requirement the substrate that fixes is cut into required bar, obtains described pork measles gold test strip bar.

8, claim 6 or 7 described pork measles gold test strip bar preparation methods is characterized in that also being provided with the whole blood filtration unit on the application of sample water accepting layer.

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