Construction Of RNA Interference Vector With Homologous Recombination Ability To Swine Genome And Cell Model Resisting FMDV Infeection

RNA interference is to inhibit the gene expression by small RNAs (miRNA, siRNA, and shRNA).RNAi gradually instead the use of targeting vector, and was becoming an important gene function studytool. miRNA and siRNA were used to transiently inhibit the gene expression. As shRNA can be stableexpressed under the control of human U6promoter and terminator, it can be used to construct cell linewith stable expression function. The delivery systems used to deliver the hU6expression element wereplasmid with selection marker and virus vector (Herpesviruses, Lentivirus, Poxviuses, and Adenovirus).The integration efficiency of plasmid was low, and the integration site was random. Virus vectorswere with high integration ability; however its random integration takes difficulty to genome locationwhich is important to the confirmation of a cell line or a transgenic animal species. What is more, somethink virus vector maybe a potential threaten to public health. As those problems, targeting vector wasbecoming a topic as the delivery system of RNAi. As the widely use of RNAi and the more clear genefunction study, it was possible to interfere the gene function of the pathogen or host. In this research, thetargeting vector and RNAi were combined in use which would favour the generation of transgenicanimals.A targeting vector homologous to the third intron region of β3gene with the hU6expressionelement to αv was constructed. From the swine genome, homologous armes were amplifed while thehU6expression element was cloned from plasmid iαv-pENTR/U6. Through several steps of subclone,the targeting vector pSN-U6with hU6expression element was constructed. After pSN-U6waslinearized, it was electrotransfection to PK-15cells. After selection with G418and PCR identification, acorrectly recombinant cell strain was screened. Gene activity of β3and αv was further detected byq-PCR, while the protein expression of αv on cell surface was detected by Cell Indirect-ELISA.Statistics showed that there was no obvious change for mRNA level of β3, while the mRNA andproteins level were obviously declined. Viral challenge and TCID50detected were all show a resistantto FMDV for the positive monoclonal cell.