Recombinant protein E2 and application thereof
阅读说明：本技术 一种重组蛋白e2及其应用 (Recombinant protein E2 and application thereof ) 是由 盛金良 肖盛中 李岩 马旭升 杨艳 张彦红 张哲� 陈创夫 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种重组蛋白E2,具有序列表SEQ.ID.No.1的氨基酸序列；所述重组蛋白E2具有序列表SEQ.ID.No2碱基序列,还提供了应用,用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒。本发明E2重组蛋白准确率高,既保证了蛋白的中和性,又避免了高突变率的困扰,更利于运用在BVDV抗体检测技术的开发。E2重组蛋白使用传统的蛋白原核表达技术,成本低,表达量大,成功表达出的E2重组蛋白,经尿素梯度复性后,West blot检测反应原性强。该E2重组蛋白用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,建立的BVDV抗体ELISA检测方法灵敏度高,特异性好,更利于在基层广大农户中推广使用,更利于我国牛群中BVDV的净化。(The invention provides a recombinant protein E2, which has an amino acid sequence of a sequence table SEQ.ID.No.1; the recombinant protein E2 has a base sequence of SEQ ID No.2 in a sequence table, and also provides application of the recombinant protein E2 in an indirect ELISA kit for detecting bovine viral diarrhea virus antibodies. The E2 recombinant protein has high accuracy, ensures the neutralization of the protein, avoids the trouble of high mutation rate, and is more beneficial to the development of BVDV antibody detection technology. The E2 recombinant protein uses the traditional protein prokaryotic expression technology, the cost is low, the expression quantity is large, the successfully expressed E2 recombinant protein has strong reactogenicity in West blot detection after urea gradient renaturation. The indirect ELISA kit for detecting the bovine viral diarrhea virus antibody by using the E2 recombinant protein has the advantages of high sensitivity and good specificity of the established BVDV antibody ELISA detection method, is more beneficial to popularization and use in primary farmers and is more beneficial to purification of BVDV in cattle flocks in China.)
1. A recombinant protein E2, wherein the recombinant protein E2 has an amino acid sequence of SEQ ID No.1 of the sequence list; the recombinant protein E2 has a nucleotide sequence of a sequence table SEQ.ID.No2; the preparation method of the recombinant protein E2 comprises the following steps: optimizing an E2 protein amino acid sequence, adopting whole gene synthesis, inserting a recombinant E2 protein gene into an expression vector pET30a through restriction enzyme cleavage sites NdeI and HindIII, confirming the accuracy of the final expression vector through an enzyme cleavage method and sequencing, finally sequentially transferring into a Top10 escherichia coli clone strain competent cell and a BL21(DE3) escherichia coli expression strain competent cell, inducing and expressing a recombinant protein E2 through IPTG, and then purifying through Ni-IDA affinity chromatography to obtain the recombinant protein E2 with biological activity.
2. The use of the recombinant protein E2 as claimed in claim 1, wherein the recombinant protein E2 is used in an indirect ELISA kit for detecting bovine viral diarrhea virus antibodies, and the indirect ELISA kit comprises a coated ELISA plate, negative control serum, positive control serum, an enzyme-labeled secondary antibody diluent, a concentrated washing solution, a sample diluent, a color developing solution and a stop solution, wherein the coated ELISA plate uses the recombinant protein E2 as a coating antigen.
3. The use of claim 2, wherein said coated microplate is coated with 0.2 μ g of said recombinant protein E2 per well;
the negative control serum and the positive control serum are respectively 2 mL;
the enzyme-labeled secondary antibody is obtained by diluting rabbit anti-bovine IgG by 100 times with HRP coupled substance stabilizing/diluting agent I, and the enzyme-labeled secondary antibody is 0.6 mL;
the enzyme-labeled secondary antibody diluent is a 0.01M phosphate buffer saline solution b containing pH7.2-7.4, the mass fraction of horse serum in the phosphate buffer saline solution b is 5%, and the enzyme-labeled secondary antibody diluent is 60 mL;
the concentrated washing solution is 20 multiplied by the washing solution, the concentrated washing solution is 0.1M phosphate buffer salt solution c with the pH value of 7.2-7.4, the mass fraction of Tween 20 in the phosphate buffer salt solution c is 0.5%, and the concentrated washing solution is 100 mL;
the sample diluent is a phosphate buffer saline solution d with the pH value of 7.2-7.4 and the mass fraction of 0.01M, the mass fraction of bovine serum albumin in the phosphate buffer saline solution d is 0.1%, the mass fraction of sucrose in the phosphate buffer saline solution d is 2%, and the sample diluent is 120 mL;
the color developing solution comprises a color developing solution A and a color developing solution B, and the preparation method of the color developing solution A comprises the following steps: dissolving 200mg of tetramethylbenzidine in 100mL of absolute ethanol, and diluting the solution to 1000mL with double distilled water to obtain a color developing solution A; the preparation method of the color developing solution B comprises the following steps: weighing 21g of citric acid and 28.2g of anhydrous sodium phosphate, adding 6.4mL of 0.75% urea hydrogen peroxide solution, diluting double distilled water to 1000mL, and adjusting the pH value to 4.5-5.0 to obtain a color development liquid B; the color developing solution A and the color developing solution B are both 30 mL;
the stop solution is 2M H2SO4The solution is 100 mL.
Technical Field
The invention belongs to the technical field of gene recombination, and particularly relates to a recombinant protein E2 and application thereof.
Background
Bovine viral diarrhea/mucosal disease (BVDV/MD) is an important infectious disease of pigs, cattle, sheep, camels and other wild animals caused by Bovine Viral Diarrhea Virus (BVDV), and has high incidence and low mortality. Clinical types include acute infections such as pneumonia, diarrhea, female animal abortion, dead fetus and teratocarcinosis, mucosal diseases and persistent infections, and secondary infection caused by immunosuppression and failure of other vaccine immunity. Persistent Infection (PI) is also a form of BVDV maintenance in its natural environment, primarily caused by intrauterine infection at the early stages of pregnancy in cows. At the moment, the immune system of the fetus does not mature, and can not identify external non-cytopathic BVDV, so that the fetus is continuously infected after birth and is in an immune tolerance state, and specific antibodies are not generated in vivo, but are toxic and toxin-expelling for the whole life. Persistently infected cattle are stores of BVDV in cattle farms, and sick cattle can discharge 100 to 1000 ten thousand of virus particles into the environment through excrement such as feces every day, which is the most main infection source for BVDV diffusion. With the promotion of large-scale cultivation, the spread of BVDV is accelerated, and the infection condition of domestic BVDV is increasingly aggravated. At present, BVDV has no effective cure mode temporarily, and the breeding process mainly depends on the purification, prevention and control of cattle. Therefore, the BVDV antibody detection method which is low in cost, convenient, simple, rapid, effective and beneficial to popularization is established, and the cattle with diseases and recessive infection can be detected and eliminated in time, so that the healthy development of the cattle group is better ensured.
The E2 protein is located on the surface of an envelope of a BVDV virus particle, is a research hotspot of pestiviruses at home and abroad at present, is located at 2077-3198 nucleotides of ORF and consists of 374 amino acid residues, and the molecular weight of the non-glycosylated protein is about 42 kDa. The C end of the E2 protein contains a hydrophobic membrane anchoring area, and the hydrophobic membrane anchoring area is anchored on the surface of the envelope, so that the infected animal can be induced to produce virus neutralizing antibodies, the body is stimulated to generate immune response, and the immune response is simultaneously involved in the recognition and adsorption of cells. The half of E2 near the N-terminus contains multiple conformation-dependent antigenic domains, the N-terminus of which protrudes beyond the surface of the envelope of BVDV and is the primary site responsible for the antigenicity of BVDV, as well as binding to anti-BVDV antibodies, mediating the immune neutralization reaction, and recognition and adsorption by the host cell. The E2 protein has neutralizing activity, but has the characteristic of high mutation. Therefore, when the E2 protein is selected as a diagnostic antigen, the conserved regions of the E2 protein of different epidemic strains of BVDV need to be analyzed, the regions which are conserved, stable and representative in the E2 protein and contain dominant antigen epitopes are selected and inserted into different vectors, so that the high-efficiency expression of the E2 recombinant protein is realized, and the method plays an important role in the development of BVDV antibody diagnostic technology and the prevention and control of diseases.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a recombinant protein E2 and application thereof aiming at the defects of the prior art, the E2 recombinant protein has high accuracy, the neutralization of the protein is ensured, the trouble of high mutation rate is avoided, and the development of the BVDV antibody detection technology is facilitated. The E2 recombinant protein uses the traditional protein prokaryotic expression technology, the cost is low, the expression quantity is large, the successfully expressed E2 recombinant protein has strong reactogenicity in West blot detection after urea gradient renaturation. The indirect ELISA kit for detecting the bovine viral diarrhea virus antibody by using the E2 recombinant protein has the advantages of high sensitivity and good specificity of the established ELISA detection method for the BVDV antibody, and is more favorable for popularization and use in vast farmers at the basic level.
In order to solve the technical problems, the invention adopts the technical scheme that: a recombinant protein E2, wherein the recombinant protein E2 has an amino acid sequence shown in a sequence table SEQ.ID.No. 1; the recombinant protein E2 has a nucleotide sequence of a sequence table SEQ.ID.No2;
the preparation method of the recombinant protein E2 comprises the following steps: optimizing an E2 protein amino acid sequence, adopting whole gene synthesis, inserting a recombinant E2 protein gene into an expression vector pET30a through restriction enzyme sites Nde I and Hind III, confirming the accuracy of the final expression vector through an enzyme cutting method and sequencing, finally sequentially transferring into a Top10 escherichia coli clone strain competent cell and a BL21(DE3) escherichia coli expression strain competent cell, inducing and expressing a recombinant protein E2 through IPTG, and then purifying through Ni-IDA affinity chromatography to obtain a recombinant protein E2 with biological activity;
the BL21(DE3) Escherichia coli is a strain which is used for a protein expression host of T7 RNA polymerase which is a high-efficiency exogenous gene of an expression system, the expression of the T7 phage RNA polymerase gene is controlled by a lacUV5 promoter of a phage DE3 region, the region is integrated on a chromosome of BL21, and the strain is suitable for the expression of non-toxic protein.
The invention also provides application of the recombinant protein E2, wherein the recombinant protein E2 is an indirect ELISA kit for detecting bovine viral diarrhea virus antibodies, the indirect ELISA kit comprises a coated ELISA plate, negative control serum, positive control serum, an enzyme-labeled secondary antibody diluent, a concentrated washing solution, a sample diluent, a developing solution and a stop solution, and the coated ELISA plate takes the recombinant protein E2 as a coating antigen.
Preferably, each well of the coated ELISA plate is coated with 0.2 mu g of the recombinant protein E2;
the negative control serum and the positive control serum are respectively 2 mL;
the enzyme-labeled secondary antibody is obtained by diluting rabbit anti-bovine IgG by 100 times with HRP coupled substance stabilizing/diluting agent I, and the enzyme-labeled secondary antibody is 0.6 mL;
the enzyme-labeled secondary antibody diluent is a 0.01M phosphate buffer saline solution b containing pH7.2-7.4, the mass fraction of horse serum in the phosphate buffer saline solution b is 5%, and the enzyme-labeled secondary antibody diluent is 60 mL;
the concentrated washing solution is 20 multiplied by the washing solution, the concentrated washing solution is 0.1M phosphate buffer salt solution c with the pH value of 7.2-7.4, the mass fraction of Tween 20 in the phosphate buffer salt solution c is 0.5%, and the concentrated washing solution is 100 mL;
the sample diluent is a phosphate buffer saline solution d with the pH value of 7.2-7.4 and the mass fraction of 0.01M, the mass fraction of bovine serum albumin in the phosphate buffer saline solution d is 0.1%, the mass fraction of sucrose in the phosphate buffer saline solution d is 2%, and the sample diluent is 120 mL;
the color developing solution comprises a color developing solution A and a color developing solution B, and the preparation method of the color developing solution A comprises the following steps: dissolving 200mg of tetramethylbenzidine in 100mL of absolute ethanol, and diluting the solution to 1000mL with double distilled water to obtain a color developing solution A; the preparation method of the color developing solution B comprises the following steps: weighing 21g of citric acid and 28.2g of anhydrous sodium phosphate, adding 6.4mL of 0.75% urea hydrogen peroxide solution, diluting double distilled water to 1000mL, and adjusting the pH value to 4.5-5.0 to obtain a color development liquid B; the color developing solution A and the color developing solution B are both 30 mL;
the stop solution is 2M H2SO4The solution is 100 mL.
Compared with the prior art, the invention has the following advantages:
the E2 protein in the BVDV has the characteristics of strong neutralization activity and high mutation rate. Combining with E2 protein has strong reactogenicity, analyzing the base sequence composition of the conserved part of the E2 protein of the epidemic BVDV strain, selecting the part with antigen dominant epitope in the conserved part, and obtaining the target fragment by artificial synthesis. Compared with the traditional truncated expression of the dominant epitope of the E2 protein antigen, the artificially synthesized and expressed E2 recombinant protein has high accuracy, ensures the neutralization of the protein, avoids the trouble of high mutation rate, and is more beneficial to the development of BVDV antibody detection technology. On the other hand, the E2 recombinant protein uses the traditional protein prokaryotic expression technology, the cost is low, and the expression quantity is large. After urea gradient renaturation, the Westblot detection reactogenicity of the E2 recombinant protein which is successfully expressed is strong. The indirect ELISA kit for detecting the bovine viral diarrhea virus antibody by using the E2 recombinant protein has the advantages of high sensitivity and good specificity of the established BVDV antibody ELISA detection method, is more beneficial to popularization and use in primary farmers and is more beneficial to purification of BVDV in cattle flocks in China.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a graph showing the accuracy of the restriction analysis of prokaryotic expression plasmid pET30a-E2 in example 1 of the present invention.
FIG. 2 is a graph showing the IPTG-induced expression of the recombinant protein E2 expression strain identified by SDS-PAGE analysis in example 1 of the present invention.
FIG. 3 is a diagram showing the results of SDS-PAGE analysis of supernatant c of a bacterial liquid obtained by inducing and then crushing and removing impurities from a recombinant protein E2-expressing strain in example 1 of the present invention.
FIG. 4 is a diagram showing the result of purifying inclusion body precipitate b after induction and disruption of recombinant protein E2 expressing bacteria by SDS-PAGE in example 1 of the present invention.
FIG. 5 is a SDS-PAGE result of the purified recombinant protein E2 of example 1 of the present invention.
FIG. 6 shows a Western-blot analysis of the purified recombinant protein E2 of example 1 of the present invention.
Detailed Description