A Preliminary Study On The TRIM5alpha Gene Editing Of The Macaca Fascicularis

AIDS is a major human disease, although a large number of exploratory studies have been carried out at home and abroad, there is no effective means of prevention and control, one of the main reasons is the lack of animal disease models can be infected with HIV. At present, many studies have found that TRIM5α is a limiting factor to inhibit HIV, can inhibit a variety of retroviral, it is an important antiviral factor.TRIM5α contains RING domain, B-Box domain, Coiled-coil domain and SPRY(B32.0) domain. HIV envelope and host cell membrane fusion, the capsid and TRIM5α protein multi poly specific binding, with the help of TRIM5α ubiquitin ligase activity and interferes with the virus uncoating of the orderly process, so as to achieve the purpose to suppress the virus. TRIM5α exists in the cell mainly in the form of trimer, and the intracellular TRIM5α protein has the ability to inhibit HIV-1. The TRIM5α of the macaca fascicularis has a stronger inhibitory effect on HIV-1 than human TRIM5α. In this study, we tried to establish the animal model of HIV and the important function of TRIM5α by knocking down the TRIM5α gene of macaca fascicularis.Based on this, this study in cynomolgus monkey gene TRIM5α, using RNAi Technology(ribose nucleic acid interfere) synthesis of the corresponding sh RNA(short hairpin RNA) and construct the corresponding sh RNA sequences of three sh RNA plasmid containing, respectively named sh RNA1 plasmid, sh RNA2 plasmids and sh RNA3 plasmid. 293 T cells were co transfected with expression plasmids and packaging plasmids to produce the corresponding lentivirus vectors. Use lentivirus vectors infected cynomolgus monkey embryonic fibroblast cells(Cynomolgus monkey embryo fibroblast, CMEF), infected cells by flow cytometric sorting stable expression of CMEF cell line, were named as NTC(negative temperature coefficient)-CMEF, sh RNA1-CMEF, sh RNA2-CMEF and sh RNA3-CMEF cells. By QRT PCR to detect the expression level of TRIM5α gene, it was found NTC-CMEF intracellular TRIM5α gene level is the expression level of wild-type CMEF 97%, sh RNA1-CMEF, sh RNA2 and sh RNA3 cells within the TRIM5α gene level are 52% of the wild-type CMEF, 43% and 39%. By Western blot TRIM5α protein expression levels found NTC-CMEF intracellular protein TRIM5α expression level is 108% of the expression level of wild-type CMEF. Sh RNA1-CMEF, sh RNA2-CMEF and sh RNA3-CMEF intracellular TRIM5α gene and protein level respectively, 59% of the wild-type CMEF, 47% and 49%. In the validation TRIM5α knock low of HIV infection, the RFP expressing fake HIV, respectively, the NTC-CMEF, sh RNA1-CMEF, sh RNA2-CMEF and sh RNA3-CMEF cells again infection experiments. The results show that TRIM5α gene knockdown macaca fascicularis cell lines relative to that of wild type cells can inhibit the pseudotyped HIV-1 virus’ s ability to infect. The infection ability of sh RNA3 lentivirus vector was preliminarily verified in the embryo of the cynomolgus monkey.To sum up, the present study was successfully obtained lentivirus vector at the cellular level to be verified by the TRIM5α gene knockout of cynomolgus monkey, and in the cynomolgus monkey embryos were carried out a preliminary study. The research of this paper provides important ideas and basis for the establishment of animal model of AIDS disease.