| Bovine virus diarrhea mucosal disease(BVD-MD), mainly caused by cloven hoofed animals, complications of infection, cause huge economic losses to the livestock breeding industry in China. Bovine viral diarrhea virus(BVDV) spread widely in the world. The main clinical symptoms showed diarrhea, acute and chronic mucosal disease and persistent infection and immune tolerance, mainly caused by the dam of abortion, stillbirth and fetal malformation. Xinjiang is the main production base of animal husbandry in China, and it is also one of the main areas of the disease. However, there is no effective treatment for the disease. Therefore, the disease has brought great security risks and economic losses to the livestock breeding industry and the food industry. Scientists in the United States found single domain antibody in camel opened a new chapter in the cloned antibody; it is lack of light chain of the antibody, because of its small volume, also known as nanobody. Nanobody has many advantages, such as high temperature resistance, easy to be expressed in the prokaryotic system, good affinity, and strong tissue penetration and so on.Objective: The use of phage display technology from the anti BVDV nanobody gene library screening. Get high efficiency and high specificity of nanobodies, Lay the foundation for research and development of biological agents to prevent and control BVDV.Methods:(1) The whole sequence of BVDV-E2 gene of bovine viral diarrhea virus was analyzed, which was found to contain a large number of rare cordon and hydrophobic region. At the same time, on the basis of BVDV-E2 gene encoding protein antigen table for analysis and prediction;(2) Using phage display technology, build the BVDV nanobody library of helper phage m13k07 to save and enlarge it, after rescue and amplification of the library. Take three rounds of affinity selection and screening of specificity monoclonal;(3) The successful analysis of sequence for primer design, and use PCR technique of gene fragments were amplified and to construct the prokaryotic expression vector of p EGX-4T--VHH. By SDS-PAGE experiment for the purpose of protein were detected by Ni column will express the protein was purified and protein of dialysis and concentration;(4) Use the double sandwich ELISA validation of nanobody binding and neutralization tests for the virus infected MDBK cells, and establish the negative control group and positive control group.Results:(1) Found intensive 630 bp antigen epitope regions in BVDV-E2, selects the region fragment in Escherichia coli for expression, purification and functional verification, the display to obtain the expected antigen protein;(2) BVDV-E2 nanobody screening positive monoclonal. And sequence positive clone, analyze the sequence is belong to camel heavy chain antibody fragments;(3) BVDV-E2 protein nanobodies in prokaryotic system successfully expressed gain size 49.5KD of the protein and purified;(4) nanobody virus neutralization function verification results show nanobody protein can neutralize the virus so as to protect the MDBK cells will not be damaged.Conclusion: This experiment get BVDV-E2 epitope protein from by prokaryotic system structure built of antibody library screening specific antibodies directed against the BVDV-E2, the nanobody to BVDV neutralizing, in a certain extent protect MDBK cells to avoid the occurrence of lesions and laid a very important foundation for the development and study of BVDV treatment and prevention. |
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