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Cloning and Expression gp90 Gene of Reticuloendotheliosis Virus (REV) and Development of Indirect-ELISA for Detection of Antibody to REV

Author: YuChong
Tutor: HuangYong
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Reticuloendothelial hyperplasia virus isolates in Sichuan gp90 gene The prokaryotic expression ELISA method
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 45
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Abstract

The avian reticuloendothelial hyperplasia is a kind of avian reticuloendothelial hyperplasia virus by the retrovirus family (reticuloendotheliosis virus, REV) chickens, ducks, geese and other birds caused by pathological syndrome, REV infection can cause tumor growth stunting syndrome, gastritis, infected thymus and cavity capsule and other immune organs, causing tissue and organ atrophy, reduce the poultry immunity even lead to immune suppression, great for viral and bacterial Following infection provides convenience. RE According to reports in China's fashion trends have a tendency to expand its poultry infection in China has reached 20% to 30%, the major significance of REV detection prevention. By cloning, expressing the the REV immunodominant protein, the study was designed to establish a fast and accurate ELISA detection method. Avian reticuloendothelial hyperplasia virus (reticuloendotheliosis virus REV) SNV strains before cDNA sequence of the genome, bioinformatics software analysis found that the gp90 gene antigen activity across high hydrophobicity, is not conducive to the smooth progress of the prokaryotic expression . Designed and synthetic primer ends to add the restriction endonuclease enzyme sites EcoR I and Hind III, as a template. Isolates REV-SC1 amplified the REV capsular gene gp90 antigen, hydrophilic high 121 ~ 1023 bp gene fragments, avoiding both ends of the low antigenicity strong hydrophobic region; the purified target gene fragment, the target gene to pMD-19T vector to construct the recombinant cloning vector pMD-19T-gp90 bacteria DH5α was transformed into E. coli Engineering, PCR and double digestion of recombinant cloning plasmid sequencing positive clones were recombinants sent to biotech companies, stitching feedback sequence comparison and analysis; use restriction endonuclease enzyme sites EcoR Ⅰ, Hind III recombinant cloning vector pMD-19T-gp90 and prokaryotic expression vector pET-32a () double digestion, purification recycling the gp90 gene fragment and the linearized plasmid pET-32a (), the gene fragments in the correct reading frame was cloned into pET-32a () , build reorganization prokaryotic expression plasmid pET-32a ()-gp90, was transformed into E. coli BL21 (DE3), the identification of positive recombinants using PCR and restriction enzyme digestion, sequencing of biotech companies rushed identification is correct, careful validation of sequencing results efficient than cloning sequencing results, focusing on checking whether a gene mutation; recombinant Escherichia coli with IPTG induced expression by SDS-PAGE and Western-blot methods were elucidated expression product size and antigenicity of the expression product of the bacteria The major form of identification. Inclusion body washing, purification, renaturation, to investigate recombinant gp90 protein coating antigen initially established indirect ELISA method for detection REV. The results show that: 1) successfully amplified the gp90 gene sequencing results showed that the recombinant cloning vector pMD-19T-gp90 gp90 nucleotide sequences in GenBank REV-SC1 strain has published the highest sequence homology of 99% at 231, 870 occurred at a two-base mutation Phylogenetic analysis showed that, with the Heilongjiang isolates ZD0708 JLR0903 closest genetic relatives; 2) SDS-PAGE electrophoresis identification of expressed protein of about 45 Kd, in line with the expected molecular size, Western-blot The results showed that the biologically active recombinant protein expression products mainly in the form of inclusion bodies in bacterial internal variability, washing, refolding operation of inclusion bodies, the pure target protein; 3) ELISA detection method: Best antigen a dilution of 1:20 (for a concentration of about 1.5 mg / ml), an anti-dilution of 1:320, positive sera to determine the critical value of 0.126, specificity, sensitivity and reproducibility of test results. The trial to explore the cloning and expression of the REV immunodominant gp90 protein, and its application ELISA test was positive and beneficial to explore its commercialization, has laid a solid foundation for large-scale applications.

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