CN112345768B

CN112345768B - A kind of indirect ELISA detection method and kit of livestock chlamydia antibody based on recombinant protein rsPgp3

Info

Publication number: CN112345768B
Application number: CN202011155394.XA
Authority: CN
Inventors: 胡思顺; 周文; 胡长敏; 舒晓倩; 袁李圣兰; 李自力; 刘梅
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-05-07
Anticipated expiration: 2040-10-26
Also published as: CN112345768A

Abstract

The invention belongs to the technical field of biomedical detection, and particularly relates to an indirect ELISA detection method and a kit for a livestock chlamydia antibody based on a recombinant protein rsPgp3(rsPGP 3). The invention obtains the livestock chlamydia rsPgp3 by gene cloning expression technology. An indirect ELISA method is established by taking rsPgp3 as a coating antigen, and comprises the preparation of the antigen rsPgp3, the establishment of indirect ELISA, the establishment of a judgment standard and the application of clinical serological detection. The method can be used for detecting the specific antibody of the livestock chlamydia Pgp3 protein, so as to judge the chlamydia morbidity and the natural infection level in the cattle and sheep. At present, the method is to establish ELISA for detecting the chlamydia of cattle and sheep by using livestock chlamydia Pgp3 protein for the first time in China. The secondary antibody adopts streptococcus G protein (HRP-SPG) marked by HRP, and the specificity and the sensitivity of the secondary antibody are obviously higher than those of the traditional secondary antibody.

Description

Indirect ELISA detection method and kit for livestock chlamydia antibody based on recombinant protein rsPgp3

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to an indirect ELISA detection method and a kit for a livestock chlamydia antibody based on a recombinant protein rsPgp 3.

Background

Chlamydia is a unicellular prokaryotic microorganism parasitized in obligate cells between bacteria and viruses, has a unique growth and development cycle, can cause chlamydiosis of human and animals, causes various inflammations, can influence a reproductive system, and causes a great public health problem. The chlamydia has wide hosts, and pigs, cattle and sheep in livestock are susceptible to the chlamydia, so that the chlamydia has no obvious age difference, but the symptoms of the livestock and poultry with different ages after infection are different. In recent years, the infection of livestock chlamydia is very common in Chinese farms, and can cause various diseases such as abortion, stillbirth and the like of female animals, thereby bringing huge economic loss to the breeding industry. The traditional chlamydia laboratory diagnosis method has the defects of long detection time, high requirements on operators and instruments and equipment, and limited detection sensitivity, which causes missed detection, and the serological diagnosis method mainly comprises complement fixation experiments, forward indirect hemagglutination experiments and ELISA, and the complement fixation experiments are complex in operation and not suitable for popularization. The indirect hemagglutination test is simple to operate, but has poor sensitivity. The chlamydia proteins currently used for antigen diagnosis include outer membrane protein ompA and macrophage infection enhancing protein (MIP), but both have reasons of insufficient specificity or strong cross-reactivity and the like, and cannot be popularized and commercialized. According to the existing domestic literature reports that the positive rate of animal chlamydia infection in domestic farms is high and the animal chlamydia infection tends to increase year by year in certain provinces, and in order to better diagnose and control the domestic animal chlamydia disease, a rapid and accurate chlamydia diagnosis method is urgently needed to be established. According to the report of related documents, the Pgp3 protein of the chlamydia plasmid is highly conserved, has species specificity and good antigenicity. The detection of Liudongming (2015) on the serum anti-chlamydia immunodominant protein antibody of a person infected with chlamydia trachomatis in the genitourinary tract is analyzed, and the detection result shows that the pathogen plasmid coded protein 3 is one of chlamydia proteins with strong antigenicity and high detection rate (72%). However, Pgp3 has not been noted and used in animal chlamydia.

At present, the serological monitoring of chlamydia infection in China mainly depends on a chlamydia indirect hemagglutination IHA detection kit developed by Lanzhou veterinary institute, and has the defects of more time consumption, sensitivity to be improved and the like, so that a rapid and accurate chlamydia diagnosis method is urgently needed to be established. Enzyme-linked immunosorbent assay (ELISA) has the advantages of simple operation, high sensitivity, high specificity, rapidness, easy standardization and the like, and is suitable for large-scale serological detection.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an indirect ELISA detection method and a kit for a livestock chlamydia antibody based on a recombinant protein rsPgp3, and aims to solve part of the problems in the prior art or at least alleviate part of the problems in the prior art.

The invention is realized in such a way that an indirect ELISA detection method for the non-diagnosis purpose of the livestock chlamydia antibody based on the recombinant protein rsPgp3 comprises the following steps:

diluting the recombinant protein antigen recombinant protein rsPgp3 with a coating solution, coating the diluted recombinant protein rsPgp3 on a 96-well enzyme label plate at 100 mu L/well, and standing overnight at 4 ℃; the amino acid sequence of the recombinant protein rsPgp3 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2 (the sequence is derived from a livestock chlamydia sequence);

throwing off the coating liquid, and washing the plate for 3 times with washing liquid for 5 minutes each time;

adding 100 mu L/hole sealing liquid, sealing for 2h at 37 ℃, taking out and washing the plate;

diluting the serum sample by PBST, repeating the steps for 100 mu L/hole, reacting at 37 ℃ for 1h, and taking out and washing the plate;

adding HRP-SPG, reacting at the temperature of 37 ℃ for 1h in a hole of 100 mu L, and taking out the plate;

adding TMB substrate solution, reacting for 10-15min in dark place, adding stop solution 2M H₂SO₄50 μ L/well, stop the reaction and read the OD immediately on the microplate reader₄₅₀The absorbance value of each hole at nm is judged as positive result when OD450nm is more than 0.23, and is judged as negative when OD450nm is less than or equal to 0.23.

Preparation of coating antigen: the DNA sequence (GenBank: KT352923.1) of the plasmid protein Pgp3 coded by the chlamydia of livestock (cattle and sheep) is searched according to GenBank, the epitope of the coded protein is analyzed according to DNAstar biological software, the main antigen region 66aa-264aa is screened, and the corresponding DNA fragment is synthesized by Tianyihui Yuan biology Limited company and is connected to a pET-28a (+) vector to construct a recombinant plasmid pET28a-spgp3 after codon optimization. pET28a-spgp3 is transformed into BL21(DE3), rsPgp3 is efficiently expressed by induction of 1mM IPTG, the protein is mainly expressed in a supernatant form, part of the protein is expressed in an inclusion body, and a recombinant protein rsPgp3 is obtained by purification through a Ni + affinity chromatography method.

Further, the coating solution was 0.05M carbonate buffer at pH 9.6.

Further, the dilution of the recombinant protein antigen was 8. mu.g/mL.

Further, the dilutions of serum samples were 1: 200.

further, HRP-SPG was diluted with PBST at a dilution of 1: 8000.

an indirect ELISA detection kit for livestock chlamydia antibody based on recombinant protein rsPgp3, comprising: recombinant protein rsPgp3, buffer solution, confining liquid, sample diluent, enzyme-labeled antibody HRP-SPG, developing solution and stop solution; the amino acid sequence of the recombinant protein rsPgp3 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO. 2. The sequence of the recombinant protein rsPgp3 in the invention is derived from the sequence of cattle and sheep chlamydophila, and is obtained by taking escherichia coli as an expression vector of a target protein for expression.

Further, the blocking solution was PBST with 2% BSA.

Further, the sample diluent was PBST supplemented with 2.5% e.

In summary, the advantages and positive effects of the invention are:

the Pgp3 protein for detecting the livestock chlamydia antibody selects a main antigen region (66aa-264aa) of Pgp3 protein in a livestock chlamydia plasmid as a target antigen, and uses a prokaryotic expression vector pET-28a (+) and a receptor bacterium BL21(DE3) to efficiently express pET28a-spgp3 to obtain a recombinant protein rsPgp 3. The protein is mainly expressed in a supernatant form, part of the protein is expressed by inclusion bodies, and the truncated recombinant protein Pgp3 is obtained by purifying the protein by a Ni + affinity chromatography method. The sPGP3 recombinant protein used in the invention is a purified gene expression product, but not a live chlamydia whole thallus, thereby avoiding the potential safety hazard of infection and toxin dispersion of operators in the production process. In addition, according to the antigen epitope prediction of the Pgp3 protein by DNAstar software, the main antigen epitope is positioned at 198 amino acids at the C end, so that the specificity is obviously enhanced by carrying out truncation expression on the main antigen epitope.

Secondly, the second antibody used in the invention is a streptococcus G protein marked by HRP (HRP-SPG) for short, the streptococcus G protein is a protein in the cell wall of G, C type streptococcus, can be specifically combined with the Fc segment of an immunoglobulin IgG molecule without influencing the combination capability of the Fab segment and an antigen molecule, and has specificity and sensitivity which are obviously higher than those of the traditional second antibody. It can be combined with all subclasses of human, mouse and rat IgG, and also with IgG of guinea pig, rabbit, goat, cow, sheep and horse, etc., and is obviously superior to Staphylococcus aureus protein A which can only be combined with human and mouse IgG. The invention is not only suitable for diagnosing the chlamydia infection of the livestock, but also can be used for detecting the chlamydia diseases of other mammals, lays a foundation for the industrialized development in the future and provides powerful technical support for effectively preventing and controlling the chlamydia diseases.

And the Pgp3 antigen used in the invention takes escherichia coli as an expression vector of the target protein, the culture is simple, the quality control can be realized, and the stability is good. The method is to establish ELISA for detecting the chlamydia of cattle and sheep by using livestock chlamydia Pgp3 protein for the first time in China.

The indirect ELISA method established by the invention has the advantages of strong specificity, high sensitivity and the like, and makes up the condition that the indirect hemagglutination test judges whether negative and positive are suspicious and weak and positive are not well distinguished.

Drawings

FIG. 1 is an electrophoretogram of recombinant plasmid pET28a-spgp3 expressed in BL21(DE 3); m-protein molecular weight standard, 1-pET28a, 2-pET28a-spgp3 induction for 0h, 3-pET28a-spgp3 induction for 4h, 4-blank control, inclusion body of 5-pET28a-spgp3 lysate, and supernatant of 5-pET28a-spgp3 lysate;

FIG. 2 is a Western-blot assay of rsPgp3 recombinant protein; m-protein molecular weight standard, 1-pET28a-spgp3, 2-pET28 a;

FIG. 3 is determination of optimal working concentration of enzyme-labeled secondary antibody;

FIG. 4 is a determination of optimal conditions for coating the recombinant protein.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.

The genes, proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

The invention discloses an indirect ELISA detection method and a kit of a livestock chlamydia antibody based on a recombinant protein rsPgp3, which are shown in the following embodiments.

Example 1

1. Preparation of coating antigen

The DNA sequence (GenBank: KT352923.1) of the livestock chlamydia coding plasmid protein Pgp3 is searched according to GenBank, the antigen epitope of the coding protein is analyzed according to DNAstar biological software, a main antigen region, 66aa-264aa, a corresponding DNA fragment is synthesized by Tianyihuiyuan organism Limited company after codon optimization and is connected to a pET28a (+) vector to construct a truncated Pgp3 recombinant plasmid pET28a-spgp 3.

Induced expression and identification of recombinant protein rsPgp 3: transforming the recombinant prokaryotic expression vector pET28a-spgp3 plasmid into escherichia coli BL21 competent cells, selecting a single colony for enrichment culture, and when OD is obtained_620nmWhen the absorbance is 0.6-0.8, adding a proper amount of IPTG to induce for 5-6h, and then carrying out SDS-PAGE analysis on the bacterium. The results show that the recombinant protein rsPgp3 was successfully expressed in E.coli BL21(DE3), mainly expressed in the form of supernatant, and partially expressed in the form of inclusion body, and the protein molecular weight was 25kDa, which is substantially identical to the predicted rsPgp3 size (see FIG. 1). Reference GE HisTrap^TMThe HP purification column specification carries out rsPgp3 protein purification, and the result shows that the protein purification effect is better, and the concentration of the protein is measured to be 2 mg/mL. The operation is carried out according to the conventional Western Blotting method, and finally, an enhanced HRP-DAB substrate color development test is adoptedThe agent (purchased from Beijing Tiangen Biochemical technology company) develops color. The results show that: in contrast to SDS-PAGE, a specific band appeared at the position corresponding to Western Blotting after induction of BL21 transformed with pET28a-spgp3 (see FIG. 2). The result shows that the recombinant protein rsPgp3 can generate specific antigen-antibody reaction with antibodies in positive serum.

2. Indirect ELISA (enzyme-Linked immuno sorbent assay) established by recombinant protein rsPgp3

Determination of optimal coating concentration and optimal serum dilution of antigen rsPgp 3:

the purified rsPgp3 protein is diluted with coating solution to 2 ug/mL, 4 ug/mL, 6 ug/mL, 8 ug/mL, 10 ug/mL transverse coated enzyme label plate, 100 ul/well, each concentration coated 1 column.

② after coating, the mixture is kept at 4 ℃ overnight.

Thirdly, washing the board with washing liquid for 3 times, 5min each time, and patting the board dry on absorbent paper after washing with 300 mu L of washing liquid per hole.

And fourthly, blocking the cells by using 100 mu L/hole of 2% BSA, incubating the cells at 37 ℃ for 2h, and washing the cells.

Fifthly, the ratio of yin and yang serum is 1: 100 dilution, longitudinal multiple dilution and matrix titration to determine the optimal coating concentration of the expression product, 100 uL/well, one line of each concentration, incubation at 37 deg.C for 1h, washing and patting dry.

Adding 1: 8000 secondary antibody HRP-SPG, 100. mu.L/well, incubated at 37 ℃ for 1h, washed, patted dry.

Adding TMB substrate buffer solution 100 μ L/hole, and reacting at 37 deg.C for 10 min.

Adding 50 mul/hole stop solution to react, measuring OD by enzyme-labeling instrument₄₅₀The value of (c).

According to the results of the square matrix titration in table 1, when the dilution factor of the serum is 1:200, the positive serum OD450nm value reached 1.041 at the antigen dilution of 8. mu.g/mL, and the P/N value was also maximal. Therefore, 1:200 is the optimal serum dilution, and the optimal coating concentration of the antigen is 8 mug/mL.

Table 1 results of OD450nm values for square matrix titration

Determination of optimal working concentration of enzyme-labeled secondary antibody:

longitudinally coating the enzyme label plate by adopting the optimal coating degree and the optimal serum dilution, and diluting a secondary antibody HRP-SPG by using PBST (para-lateral peroxidase-peroxidase) in a ratio of 1:4000, 1: 6000. 1: 8000. 1: 10000, to determine the optimal working concentration of the enzyme-labeled secondary antibody.

From the results in FIG. 3, it is shown that when the enzyme-labeled secondary antibody HRP-SPG was diluted 1: at 8000, the positive and negative sera had the greatest difference in OD450nm values (P/N24), so the optimal working concentration of the enzyme-labeled secondary antibody for the recombinant protein was determined to be 1: 8000.

determination of optimal antigen coating conditions:

adding the mixture at 37 ℃ for 1h and 4 ℃ overnight; ② adding 4 ℃ overnight at 37 ℃ for 2 h; ③ 37 ℃ for 2 h; fourthly, the recombinant protein Pgp3 coated under 4 different conditions is kept overnight at 4 ℃, ELISA measurement is carried out by using bovine chlamydia disease yin-yang serum, and the optimal coating condition of the antigen is determined.

As can be seen from FIG. 4, the recombinant protein has a small difference in OD450 values between the positive serum and the negative serum under the coating conditions of 4 ℃ overnight, 37 ℃ for 1h and 4 ℃ overnight, which indicates that the coating effect is good, and for the sake of simplicity, the 4 ℃ overnight coating condition is determined as the optimal coating condition for the recombinant protein.

Determination of optimal sealing liquid and sealing time:

firstly, alum BSA; 2% BSA; ③ 5 percent BSA; and fourthly, respectively sealing the enzyme label plates by 5 percent of skimmed milk powder to determine the optimal sealing liquid. And then the enzyme label plate is respectively sealed by the optimal sealing liquid for 45min, 90min, 2h and overnight at 4 ℃. The results finally determined 2% BSA as the best blocking agent, blocking overnight at 4 ℃.

Determination of optimal reaction time of serum and enzyme-labeled secondary antibody:

the recombinant protein is used for coating an enzyme label plate under the condition of the optimal antigen coating concentration, the temperature is kept overnight at 4 ℃, the serum and the enzyme-labeled secondary antibody are both subjected to optimal dilution times, and the serum and the enzyme-labeled secondary antibody respectively react at 37 ℃ for 90min, 1h, 45min and 0.5h for ELISA determination, so that the optimal combination time of the serum and the secondary antibody is determined. Finally, the reaction time of 45min at 37 ℃ is selected as the optimal reaction time of the serum and the enzyme-labeled secondary antibody.

The ELISA operation steps are as follows:

the procedures of the ELISA assay after the above conditions were determined were as follows:

the recombinant protein antigen was diluted to an optimal concentration (8. mu.g/mL) with a coating solution (0.05M carbonate buffer at pH 9.6) and then coated on a 96-well plate at 100. mu.L/well overnight at 4 ℃. The coating solution was spun off and the plates were washed 3 times with washing solution for 5 minutes each time. Then 100. mu.L/well blocking solution (2% BSA in PBST) was added and blocked at 37 ℃ for 2h, and the plate was removed and washed as above. After diluting the serum sample to the optimal dilution (1: 200) with a sample diluent (PBST to which 2.5% of E.coli lysate was added), 100. mu.L/well was repeated in duplicate for each serum sample, and after 1 hour of reaction at 37 ℃, the plate was removed and washed as above. Then, HRP-SPG was added thereto at an optimum concentration (1: 8000) at 100. mu.L/well, reacted at 37 ℃ for 1 hour, and the plate was washed as above. Then adding TMB substrate solution, reacting for 10-15min in dark place, adding stop solution 2M H₂SO₄50 μ L/well, stop the reaction and read the OD immediately on the microplate reader₄₅₀Absorbance values at nm were measured for each well, and a blank control well was set.

Determination of ELISA negative and positive cut-off values: using the above-identified ELISA optimal conditions, 50 bovine negative sera stored in the laboratory were tested, each sample was set to 2 replicates, and OD450nm values were read, and the OD average X of all negative samples was calculated to be 0.122, the standard deviation S was calculated to be 0.036, and negative and positive results were determined according to the formula X +3S of 0.23, that is, OD450nm > 0.23 was determined as positive results, and OD450 nm. ltoreq.0.23 was determined as negative. The results are shown in Table 2 below:

TABLE 250 ELISA test results for livestock chlamydia antibody negative sera

And (3) detecting by a specificity test: ELISA cross-tests were performed using bovine foot-and-mouth disease positive serum, bovine paratuberculosis positive serum, and bovine mycoplasma positive serum under established ELISA conditions. As a result, the bovine foot-and-mouth disease positive serum, the bovine paratuberculosis positive serum and the bovine mycoplasma positive serum are all negative. The established ELISA method is shown to have better specificity. The results are shown in Table 3.

Table 3: results of specificity test

And (3) sensitivity test: livestock chlamydia positive sera were tested by established ELISA procedure and were expressed as 1: 100, 1:200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800 the dilution was done in two fold proportion and the rest conditions were performed according to the ELISA optimal reaction conditions. When the positive serum was diluted to 1: 1600 hours, the color change of the hole of the enzyme label plate is observed by naked eyes, and the negative and positive are difficult to judge, but the enzyme label can be detected, when the positive serum is diluted to 1: when 3200 hours, the result of the microplate reader detection is negative, and the result is shown in Table 4.

Table 4: results of sensitivity test

And (3) repeatability test:

(1) in-batch repeatability test: selecting 6 portions of serum which is detected to be negative, positive and suspected to be positive by IHA, diluting the serum by 1:200, carrying out repeated tests in batches on different enzyme label plates coated by the rsPgp3 purified in the same batch, setting 5 holes for each sample to be parallel, calculating standard deviation according to OD450nm values, wherein the variation coefficients are all less than 10%, and the results are shown in a table 5.

Table 5: in-batch repeat test

(2) Batch to batch repeatability test: coating the purified rsPgp3 protein of three different batches on the same ELISA plate, adding the serum to perform batch repeat test, setting 2 holes for each sample to be parallel, calculating standard deviation according to OD450nm value, wherein the variation coefficients are all less than 10%. The results are shown in Table 6.

Table 6: repeat test between lots

The results of the comparative tests are as follows: 68 parts of collected goat serum and goat serum around Wuhan are respectively detected by using an established indirect ELISA detection method and an IHA method which is universal at present in China, wherein the IHA test is performed according to the following steps of 1: and 16 are judgment holes, 4 positive parts and 64 negative parts are detected, wherein 4 serum indirect ELISA methods which are positive in IHA detection are all positive, 12 serum indirect ELISA methods which are negative in IHA detection are positive, 52 serum indirect ELISA methods which are negative in IHA detection are negative, 4 serum which are positive in both detection are obtained, 52 serum which are negative in both detection are obtained, the positive coincidence rate of the two is 100%, the negative coincidence rate of the two is 92.3%, and the total coincidence rate is 94.1%. The results are shown in Table 7.

TABLE 7 comparative test results of IHA and ELISA for bovine serum detection

And (3) stability test: and (3) coating and sealing the ELISA plate, respectively preserving at 4 ℃ and 37 ℃ for one week, detecting the yin and yang serum, and obtaining a result that the detection result of the coated ELISA plate after being preserved at 37 ℃ for one week is not obviously changed.

TABLE 8 stability test

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> indirect ELISA detection method and kit for livestock chlamydia antibody based on recombinant protein rsPgp3

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 199

<212> PRT

<213> Chlamydia

<400> 1

Thr Ile Glu Phe Ser Leu Asp Thr Pro Lys Ile Ala Gln Ala Val Leu

1 5 10 15

Glu Ser Val Thr Asn Asp Ile Met Gln Glu Ile Val Ser Asn Leu Ser

20 25 30

Gln Glu Leu Ile Glu Asp Val Leu Glu Lys Ile Asp Thr Asp Ser Ser

35 40 45

Phe Ser Phe Ser Arg Ala Phe Lys Ala Ile Glu Ile Lys Asp Cys Ile

50 55 60

Gln Cys Asn Gly Leu Phe Thr Ser Glu Asn Ile Gly Asn Leu Leu Gly

65 70 75 80

Gly Thr Glu Ile Ala Lys Phe Thr Val Thr Pro Glu Asn Ala Asn Ser

85 90 95

Ala Phe Leu Ile Asp Ala Asn Ile Ile Ala Ser Arg Met Glu Gly Ala

100 105 110

Val Val Leu Ala Leu Val Lys Glu Gly Asn Ser Ser Pro Ser Ala Ile

115 120 125

Ser Tyr Gly Phe Ser Ser Gly Leu Pro Asn Val Cys Ser Leu Lys Ala

130 135 140

Val Ile Glu Asn Thr Thr Thr Thr Pro Ala Thr Phe Ser Leu Arg Ile

145 150 155 160

Gly Gly Met Glu Ser Gly Val Val Trp Val Asn Ala Met Pro Asn Gly

165 170 175

Asn Lys Ile Leu Asn Ala Glu Thr Thr Ser Thr Ile Ser Val Leu Glu

180 185 190

Val Ile Pro Gln Thr Asn Gly

195

<210> 2

<211> 615

<212> DNA/RNA

<213> Chlamydia

<400> 2

gcgacaactc ctgcaacaat tgaattctct ctagatacac caaaaatcgc tcaagctgtt 60

ttggaaagtg ttactaatga cattatgcaa gagattgtaa gtaatttatc gcaagaattg 120

atagaagatg tgctagaaaa gattgataca gattcatctt tttccttctc tagagctttt 180

aaagctatag aaatcaagga ttgtatccaa tgtaatggac tatttacttc tgaaaatata 240

ggcaatcttc ttggaggaac agagatagca aaatttacag ttacaccaga gaacgctaat 300

agtgcatttt taattgatgc caatattata gcttctagaa tggaaggagc tgttgtctta 360

gctctggtta aagaaggaaa ctcatctcca agtgccatta gttacggttt ttcttccgga 420

ctaccgaatg tatgttccct gaaagctgtt atcgaaaata ccacaacaac tcctgcaaca 480

ttttccctaa gaataggtgg aatggaaagt ggggttgtgt gggtcaacgc aatgccaaat 540

gggaacaaaa tccttaatgc agaaacaaca tcaacaatta gtgtattaga agtaattcct 600

caaactaatg gataa 615

Claims

1. an indirect ELISA detection method based on the non-diagnostic purpose of the livestock chlamydia antibody of recombinant protein rsPgp3, is characterized in that, comprises the following steps:

The recombinant protein antigen rsPgp3 was diluted with coating solution and then coated on a 96-well microtiter plate, 100 μL/well, overnight at 4°C; the amino acid sequence of the recombinant protein antigen rsPgp3 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2 shows;

Shake off the coating solution and wash the plate 3 times with washing solution for 5 minutes each time;

Add 100 μL/well of blocking solution, block at 37°C for 2 hours, take out and wash the plate;

Serum samples were diluted with PBST, 100 μL/well, and each serum sample was repeated twice. After 1 h of reaction at 37°C, the plates were taken out and washed;

Add HRP-SPG, 100 μL/well, react at 37°C for 1 h, take out and wash the plate;

Add TMB substrate solution, react in the dark for 10-15min, add stop solution 2M H ₂ SO ₄ 50μL/well to stop the reaction, immediately read the absorbance value of each well at OD ₄₅₀ nm on a microplate reader, OD ₄₅₀ nm > 0.23 was judged as a positive result, and OD ₄₅₀ nm ≤0.23 was judged as a negative result.

2. a kind of indirect ELISA detection method based on the non-diagnostic purpose of the livestock chlamydia antibody of recombinant protein rsPgp3 according to claim 1, is characterized in that: described coating liquid is the 0.05M carbonate buffer that pH is 9.6 .

3 . The indirect ELISA detection method for non-diagnostic purposes of livestock chlamydia antibodies based on recombinant protein rsPgp3 according to claim 1 , wherein the dilution of the recombinant protein antigen is 8 μg/mL. 4 .

4 . The indirect ELISA detection method for non-diagnostic purposes of livestock chlamydia antibodies based on recombinant protein rsPgp3 according to claim 1 , wherein the dilution of the serum sample is 1:200. 5 .

5. The indirect ELISA detection method for non-diagnostic purposes of livestock chlamydia antibodies based on recombinant protein rsPgp3 according to claim 1, characterized in that: HRP-SPG is diluted with PBST, and the dilution is 1:8000.

6. an indirect ELISA detection kit based on the livestock chlamydia antibody of recombinant protein rsPgp3, is characterized in that, comprises: recombinant protein rsPgp3, buffer, blocking solution, sample dilution, enzyme-labeled antibody HRP-SPG, chromogenic solution and Stop solution; the amino acid sequence of the recombinant protein rsPgp3 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

7 . The indirect ELISA detection kit for livestock chlamydia antibodies based on recombinant protein rsPgp3 according to claim 6 , wherein the blocking solution is PBST with 2% BSA. 8 .

8 . The indirect ELISA detection kit for livestock chlamydia antibodies based on recombinant protein rsPgp3 according to claim 6 , wherein the sample diluent is PBST with 2.5% Escherichia coli lysate added. 9 .