阅读说明：本技术 一种伪狂犬病病毒ge基因主要抗原表位区域重组蛋白制备及elisa检测试剂盒 (A kind of preparation of pseudorabies virus GE gene Main Antigenic Region domain recombinant protein and ELISA detection kit ) 是由 朱传刚 周志平 冒丽 刘冀 江嘉欣 纪荣毅 沈元曦 于 2019-08-28 设计创作，主要内容包括：本发明提供一种伪狂犬病病毒GE基因主要抗原表位区域重组蛋白制备及ELISA检测试剂盒,所述蛋白为GE蛋白的核心表位,构建的gE表位原核表达的重组质粒转入Bl21后获得了高效表达,同时由于重组蛋白连接有6个His的标签,可以通过镍柱进行亲和层析,重组蛋白大小适中,又是疫苗缺失部分,因此通过间接ELISA或胶体金免疫层析试纸条法即可直接检测猪体内是否存在相关抗体,即获得猪场伪狂犬病的筛查。检测时所需的标本量极小,不需要特殊仪器,肉眼直接判读结果,且检测简便快速,特异性强,灵敏度高,准确可靠,成本低,应用广泛。(The present invention provides a kind of preparation of pseudorabies virus GE gene Main Antigenic Region domain recombinant protein and ELISA detection kit, the albumen is the core epitope of GE albumen, the recombinant plasmid of the gE epitope prokaryotic expression of building obtains high efficient expression after being transferred to Bl21, simultaneously because recombinant protein is connected with the label of 6 His, affinity chromatography can be carried out by nickel column, recombinant protein is of moderate size, it is vaccine lack part again, therefore it can directly be detected in pig body by indirect ELISA or colloidal gold immuno-chromatography test paper strip method with the presence or absence of associated antibodies, obtain the screening of pig farm pseudoabies.Required specimen amount is minimum when detection, does not need specific apparatus, the direct interpretation result of naked eyes, and detect simplicity quickly, high specificity, high sensitivity, accurately and reliably, low cost, wide application are general.)

1. a kind of ELISA kit including pseudorabies virus GE gene Main Antigenic Region domain recombinant protein, the examination It include: pseudorabies virus GE gene Main Antigenic Region domain recombinant protein, confining liquid (5% skimmed milk power), enzyme mark in agent box Secondary antibody (the rabbit-anti sheep Ig G of HRP label), PBST, developing solution (tmb substrate liquid), terminate liquid (2 mol/L sulfuric acid).

2. a kind of using pseudorabies virus GE gene Main Antigenic Region domain recombinant protein as the indirect ELISA of detection antigen Method, the method are as follows: recombinant antigen is diluted to by 4.0 μ g/ml, 2.0 μ g/ml, 1.0 μ g/ml, 0.5 μ g/ml using coating buffer With 0.25 μ g/ml, 100 holes μ l/ are coated on 96 orifice plates, and 2~8 DEG C of coatings are overnight；PBST is washed three times, and PBS is used in addition (PH7.3) diluted 5% skimmed milk power solution closing, 300 holes μ l/, 37 DEG C are closed 2 hours；PBST is washed three times, is added 1: The diluted porcine pseudorabies positive serum of 10-1:1000 and negative serum, 100 holes μ l/, 37 DEG C are incubated for 1 hour；PBST washing Three times, it is separately added into the recombination streptococcus protein G of the diluted HRP label of 1:2000 times of PBST, 100 holes μ l/, 37 DEG C are incubated for 30 points Clock；PBST is washed three times, and TMB is added and develops the color, and 100 holes μ l/, 37 DEG C are reacted 5 minutes or so；It is whole that 2mol/L H2SO4 is added It only reacts, 50 holes μ l/, reads OD450, calculate P/N value, the positive is judged to the serum of value>=2.1 P/N, with the blood of value<2.1 P/N It is judged to feminine gender clearly.

3. indirect ELISA method as claimed in claim 2, wherein the best peridium concentration of recombinant antigen is 1.0 μ g/ml, most preferably Confining liquid is the skimmed milk power solution of PBS diluted 5%, and it is 1:100 that positive serum and negative serum, which are most suitable for thinner ratio,.

4. as described in claim 1 a kind of including pseudorabies virus GE gene Main Antigenic Region domain recombinant protein ELISA kit, wherein the gene order of the recombinant protein GE is as shown in SEQ ID NO.1.

Technical field

The present invention relates to animal virologies and technical field of immunological detection, are more particularly to a kind of for detecting Swine serum The building of the viral gE albumen of middle pseudoabies antibody also relates to a kind of above-mentioned albumen of utilization and prepares immune colloid gold test paper Method, further relate to a kind of for detecting the purposes of the immune colloid gold test paper of the viral gE albumen of pseudoabies in Swine serum.

Background technique

Pseudorabies virus (Pseudorabies Virus, PRV) be cause a variety of domestic animals such as ox, sheep, pig, dog and cat and Wild animal fever, surprise itch (in addition to pig) and acute encephalomyelitis etc. for cardinal symptom herpesviral, as caused by the virus Disease symptoms claim pseudoabies like rabies.Pig is reservoir host and the infection sources of PRV, and health pig and affected pig, the malicious pig of band are straight Contact can infect the disease.Mainly cause sow breeding difficulty, miscarriage, produce the mummification of fetus, stillborn foetus and produces weak son, newborn piglet table It is now diarrhea and nervous symptoms, infection rate and the death rate are up to 100%, cause serious economic loss [1] to pig breeding industry.Disease Poison can be propagated by excretas such as body fluid, sperm, milk, excrement, can also can usually cause pig farm by air borne Seasonality outburst (Lisa E. Pomeranz et al., 2005).

The disease can occur, but multiple with winter and spring throughout the year without apparent seasonality, and virus is first in porcine respiratory Replicated (Mask, M. et al., 1965) with nasal membrane, then enter nervous system, thus cause respiratory system with Neurological symptom.Studies have shown that PRV genome is the bifilar linear DNA that size is about 150kb, 70 ~ 100 kinds can be encoded Virus protein, GE are pseudorabies virus (PRV) dispensable gene, and GE major antigen structural domain is located at GE gene the 52nd ~ 238 Between amino acid residue, the missing of GE gene can be such that the virulence of PRV substantially reduces, while GE glycoprotein can also influence PRV and exist The intracorporal tissue tropisms of pig are spread to facilitate PRV to nervous system.It is many living using gene delection since 2011 There is PR prevalence in the large-scale pig farm of vaccine, is mainly shown as that swinery GE antibody positive rate significantly increases.Therefore, GE glycoprotein It can be used as PRV Main Diagnosis antigen.Since general vaccine is g E gene-deleted strain, the pig of vaccine immunity will not generate needle To the antibody of g E albumen therefore by detecting g E antibody and can distinguish whether infected or infected PRV disease in pig farm Poison.In addition, g E albumen can promote viral transneuron to propagate and enter central nervous system and spread, in comparison g E base Because missing seedling is safer.Currently, g E Gene deletion mutation is used in combination, and by g E antibody test to infection and Immune pig carries out the effective measures that difference diagnosis is PRV removing plan.

In China, porcine pseudorabies virus gE gene-deleted vaccine is also being widely applied, and gE gene-deleted vaccine is immune After inoculation, the antibody for gE albumen cannot be detected in the Swine serum being immunized, it is wild to carry out PRV using serum Virus strain infection pig and gE gene-deleted vaccine immunity inoculation pig carry out antidiastole and provide condition, this is also porcine pseudorabies Purification and the basis for eradicating plan.Therefore, it is purifying and is eradicating in the works, the pseudorabies virus in monitoring and detection Swine serum GE protein antibodies are very important a link.Serum Antibody Detection is carried out to the pig that immunity inoculation is crossed, if detection To gE protein antibodies, illustrates that pig has infected the wild strain of porcine pseudorabies virus, the pig of infection can be eliminated, thoroughly Purification and elimination porcine pseudorabies.

Domestic detection porcine pseudorabies virus gE protein antibodies generally use enzyme-linked immunosorbent assay (ELISA) at present, But due to the particularity of this detection method, it is necessary to complete in the lab, the time used is long, costly.Therefore one is established Kind quickly, conveniently, specificity is high, sensitivity is strong, the available detection method in scene is advantageous.

Summary of the invention

It is real the invention mainly solves the technical problem of providing a kind of quick, highly sensitive immune detection product and method Now efficient, highdensity immune detection.

We have found that the 33-100 amino acids section of gE albumen n end has the antigenicity and immunogenicity of height, this One section at least has 6 epitopes, wherein 5 are all located at N-terminal extracellular region, it is residual that this section is located at 52-123 amino acids Base；Another section of epitope concentration zones are between 78 1 238 amino acids residues.Theoretically analyze, this section of extracellular region and Epitope concentration zones more likely stimulate body to generate immune response, thus are also body generation specific antibody for area Section, further, it is believed that noncontinuity epitope, i.e. conformation dependence type epitope are limited for the value of diagnosis, and connect Continuous property epitope is more suitable for diagnostic antigen detection specific antibody.For this purpose, we select the continuity of gE albumen n end anti- The amino acid sequence of former epitope, i.e. 52-238, reconstructs this section of egg according to the skewed popularity of the bacterial codon of prokaryotic expression system White gene order constructs a novel base sequence, has the gE egg of same amino acid sequence by protokaryon bacterial expression White N-terminal epitope.For the solubility for increasing expression recombinant protein, we select pET-32a (+) plasmid construction to recombinantly express Plasmid, obtains high efficient expression, and the recombinant protein expressed is present in and asks, and facilitates purifying, by the weight of affinitive layer purification Cost is relatively low as the detection method that diagnostic antigen is established for histone, and detection method is easy.Especially concentrate gE antigen main Epitope as diagnostic antigen, all more completely gE albumen is more preferable for the sensitivity and specificity of detection.

It is an object of the present invention to provide one kind to have antigenic Pseudorabies virus GE gene, using DNAstar Software analyzes amino acid sequence, theoretical molecular weight and isoelectric point expressed by recombination sequence, 5 ' BamH I of addition and 3 ' Hind III, Afterwards by the gene cloning of optimization to expression vector pET-32a (+), recombinant plasmid is obtained, is named as pET-32a (+)-rGE, GE's Gene order is as shown in SEQ ID NO.1.

Further, the present invention provides above-mentioned GE protein epitope in preparation diagnosis, prevention or treatment Pseudorabies virus diagnosis Application in reagent or drug.

In the above method, this field is can be used conventionally used for protein expression and purification in expression, the purifying of antigen-4 fusion protein gene Method, such as antigen-4 fusion protein gene is cloned into expression vector, expression vector and/or coexpression vector are transferred in expressive host It cultivates, induction vindication albumen is added after activation to logarithmic growth phase, is crushed, obtains fusion protein after purification.Wherein, of the invention Expression vector, coexpression vector, the type of expressive host and classification are not construed as limiting, this field can be selected and repaired conventionally used for heredity The carrier of decorations and host, specifically, expression vector can for pET-28, pET-32, pET-15 or pET-11 etc., coexpression vector It can be pCDFDuet-1 etc.；Expressive host can be selected from Escherichia coli, bacillus subtilis, bacillus megaterium, corynebacteria, wine Brewer yeast, Pichia pastoris or mammalian cell.

Present invention simultaneously provides a kind of product for immunoassay, the product includes fusion egg of the present invention It is white.

Further, it includes pseudorabies virus GE gene Main Antigenic Region domain recombinant protein that the product, which is a kind of, ELISA kit, include: pseudorabies virus GE gene Main Antigenic Region domain recombinant protein, envelope in the kit It closes liquid (5% skimmed milk power), ELIAS secondary antibody (the rabbit-anti sheep Ig G of HRP label), PBST, developing solution (tmb substrate liquid), terminate liquid (2 mol/L sulfuric acid).

Detailed description of the invention

The PCR of Fig. 1 recombinant plasmid identifies electrophoretogram, 1 M:DNA molecular mass standard (DL2000);A: recombination pET-32a (+)-rGE gene PCR product

The double digestion of Fig. 2 recombinant plasmid identifies electrophoretogram, M:DNA molecular mass standard (DL5000)；A: double enzyme digestion product；

The inducing expression phase of Fig. 3 GE albumen, M: protein molecular quality standard；1: non-Induction of bacterial pyrolysis product；2-7 induction Bacterial lysates after 1h, 2h, 3h, 4h, 6h, 8h；

Fig. 4 GE albumen solubility qualification result, M: protein molecular quality standard；A: recombination pET-28a (+)-rGE/BL21 (DE3 Supernatant B after transfecting bacterium: the cracking after recombination pET-28a (+)-rGE/BL21 (DE3) transfection bacterium is in the precipitating of 8M urea；

Fig. 5 GE protein immunogenic Western Blotting is as a result, M: protein molecular quality standard；A: recombinant protein.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified It is commercially available from routine biochemistry reagent shop.