Construction And Cell Transfection Of The Targeting Vectors Of Jiv90 Genes In Swine

Classical swine fever(CSF)is a highly contagious and fatal disease of pigs. Classical swine fever virus(CSFV)is a plus-strand RNA virus which is an important member of Pestivirus in Flaviviridae. CSFV could infect many kinds of cells, including T-cells, high and low density granulocytes, mononuclear cells, peripheral white blood cells, macrophagocytes and dendritic cells etc. In recent research, Jiv genes, it was found, also played extremely significant roles as molecular chaperones in host cells when CSFV infected persistently. In addition, it had already found the function of 3'-UTR of CSFV during CSFV infected.First of all, this study had used the gene targeting technology with positive-negative targeting vectors which were proved to be very effective, and it also constructed the homologous targeting vectors of Jiv90 genes from swine DNA sequences which based on the theory of gene homologous recombination via Cre enzyme and Loxp sequences, and the targeting vectors were successfully used to transfect the swine umbilical vein endothelial cells (SUVEC) in order to verify them at the cellular level preliminarily. Secondly, the vectors of 3'-UTR in CSFV for the use of RNA interference were constructed based on the study of 3'-UTR in CSFV. With these vectors, the further study of CSFV could be done.The results as follows:(1)According to the published sequences of Jiv90 genes in GenBank, the sequences of both outsides of the core area of Jiv90 genes were cloned from the extracting DNA of SUVEC as the use of long and short homologous arms in targeting vectors. In addition, the genetic fragments of the long and short arms were 4522 bp and 1944 bp respectively. After connected with pEASY-T1 vectors, the long arm vectors were cut by single enzyme HindⅢand sequencing and also the short arm vectors were cut by double enzyme ClaⅠ/SpeⅠand sequencing. Both of the results indicated that they were conformed to the requirement of homologous recombination.(2)It was successfully constructed the targeting vectors—JKS by the skeleton vectors pA2T connected with both long and short homologous arms. With transfect reagents, the JKS vectors were transfect into SUVEC. After drug screening with both G418 and GANC, it got the stable cell lines. In addition, the screening concentration of G418 was 1500μg/mL and GANC was 200 nmol/mL.(3)These cells were tested through PCR to make sure the neo genes were integrated. Then the cells were inoculated the CSFV and measured with Real-time PCR to testify their susceptibility with CSFV. The results indicated that, compared with the normal cells, the CSFV contents from the cell lines got by drug screening were remarkably deceased about 3.65 times. In a word, it is testified that the targeting vectors were successfully constructed and the Jiv90 genes were effect when CSFV infected.(4)Design and synthetic targeting shRNA sequences of three sites on 3'-UTR in CSFV. After connect with pGPU6/GFP/Neo vectors, it was testified that the three vectors were successfully constructed via sequencing. Let these vectors into PK-15 and SIEC cells by transfect reagents. Then it was got the positive cells with 85% expression of interference vectors by drug screening of G418. Through CSFV inoculated and the Real-time PCR, it was verified that the Pig1 interference vectors had better effect.