Study Of Genetic Variation Of E2 Gene Of CSFV Passaged In Porcine Testicular Cells For Many Times And Expression Of Protein E2 In The Cells

E2 gene of Classical swine fever virus (CSFV) is developed genetically engineered vaccine, which encode the gp55 protein as the major protective antigen of CSFV. The use of pig testicular cells (ST) as the receptor cells combined with the use of mammalian cell expression vector to express the E2 gene is that the first full use of receptor cells can be sub-hobby of the genetic code to improve recombinant protein expression, the other by ST from pigs, followed by processing and superiority compared to other expression hosts, their vaccine protection force and is expected to further enhance immunogenicity for genetic engineering vaccine CSFV application basis.The study included three trials. The first is that E2 gene of CSFV Fujian strain FJ1 and FJ2 were cloned and sequenced, then analysis and classical swine fever (CSF) vaccine strain C homology was done to preliminary understand the local isolates in Fujian variation. Using reverse transcription PCR (RT-PCR) and nested PCR (nPCR) method, a series of several animal passage passage swine testicular cell line E2 of classical swine fever Shimen nucleotide sequences were determined Shimen strain with the standard nucleotide and amino acid sequence homology comparison and analysis, to understand csfv in swine testicular cells after repeated passages of genetic stability. The third part is the use of pig testicular cells as receptor cells, the expression of csfv E2 gene for the establishment of the recombinant protein of classical swine fever and csfv detection method of the application of genetic engineering, the theoretical basis for the vaccine. Test and the results of this study are as follows:1. Using PCR, two Fujian popular CSF strains E2 gene were amplified, and cloned into pMD-18T vector for nucleotide sequencing. According to the Alfort strain of CSFV, Brescia strain and the C-strain (vaccine strain) to determine the starting after amino acid triplet position derived amino acid sequence, while the E2 protein homology and structural analysis were done. The results showed that, length E2 gene of both of FJ1 and FJ2 strains are 1170 bp, encoding a length of 384 amino acids, including the complete signal peptide and part of the transmembrane sequence. of 2 strains E2 gene nucleotide sequence homology is 90.79%, amino acid sequence homology of is 89.84%, with the C-strain E2 nucleotide sequence homology of 82.87%-82.96%, amino acid sequence homology of 87.76%-90.10%. The results show that in 2008 year there are some differences between E2 protein of CSF epidemic strains in parts of Fujian and C-strain virus, and this helped to lay a theoretical and physical infrastructure for current CSF prevention and control.2. CSFV Shimen strains by several passage on pig is named as P-Shimen and that by several passage on pig as ST-Shimen. Using PCR technology, P-Shimen and ST-Shimen strain E2 gene were amplified, their nucleotide were sequenced, and were compared the E2 gene with the nucleotide and amino acid sequences of P-Shimen strain, ST-Shimen strains and standard strains. The results show that P-Shimen strains and standard strains Shimen nucleotide sequence homology is 99.4%, amino acid sequence homology 99.0%, ST-Shimen strains and standard strains Shimen nucleotide sequence homology 98.3% , amino acid homology 96.7%. The results were included that ST-Shimen strain than P-Shimen E2 gene mutation was not significantly, and strains to maintain a high standard with Shimen homology showed that the CSFV in ST can be maintain its relative genetic stability, so ST provides the tools possible as CSF cell vaccine preparation.3. CSFV E2 gene were cloned from the pEGFP-C1 eukaryotic expression vector using liposome plasmid transfected into ST, then ST by fluorescent observation and G418 selection become into positive cell clones with E2 gene. Fluorescence observation and PCR analysis showed that positive cells has been cloned with E2 gene. Using the immunohistochemical test show that the E2 protein of CSFV were expressed in ST cloned with E2 gene, which lay basis for antigen detection methods by the recombinant protein of CSFV for the establishment and application of genetic engineering vaccine CSF.