Construction And Preliminary Study The Function Of Human FcγRⅡB Lentiviral Vector

Objective A lentiviral inducible expression vector for FcγRⅡB was constructed andits stable and controllable expression function was identified in HT-1080cells, then prelimin-ary study FcγRⅡB’s function in B cells.Methods Human mRNA as the template for reverse transcription to obtain FcγRⅡBgene fragment, then the FcγRⅡB gene fragment was cloned into TRE lentiviral expressionplasmid to construct TRE-FcγRⅡB lentiviral expression recombinant plasmid. TRE-FcγRⅡB lentiviral expression recombinant plasmid and Tet lentiviral inducible plasmid weretransfected into293T cells respectively with lentivirus packaging mix plasmids, to packexpression lentiviral and inducible lentiviral respectively. The viral titer of two lentiviral wasmeasured respectively. Expression lentiviral and Inducible lentiviral coinfected HT-1080cellsand induced by gradient concentration doxycycline （Dox）, then detected the expression ofFcγRⅡB by Immunofluorescence. Real-time quantitative PCR （qRT-PCR） and Westernblotting were used to detect FcγRⅡB mRNA and protein expression level. Expressionlentiviral and Inducible lentiviral coinfected Ramos cells, and induced by1000ng/mLdoxycycline （Dox）, then detected the expression of FcγRⅡB and its binding capacity forIgG Fc fragment from SLE patient’s serum IC by Immunofluorescence. This Ramos cellswere also stimulated by LPS, then use the ELISA method detects IgM secretion capacity ofRamos cells which over expression FcγRⅡB.Results The insert gene fragment of recombinant plasmid, which is identified by PCR analysis, enzyme digestion analysis and gene sequencing analysis, gene sequencing analysis’result showed that the nucleotide sequence of the inserted fragment has homology of100%with the FcγRⅡB nucleotide sequence provided by GenBank. The Expression lentiviral’svirus titer was106TU/mL and the Inducible lentiviral’s virus titer was105TU/mL. TheImmunofluorescence’s records showed that after induced by Dox, the HT-1080cells, which iscoinfected by Expression lentiviral and Inducible lentiviral, can express FcγRⅡB. The qPCRand the Western blotting’s records showed that FcγR Ⅱ B expression levels positivelycorrelated with the concentration of Dox.The Immunofluorescence’s records showed thatRamos cells can express FcγRⅡB and it’s can bind with IgG Fc fragment from SLE patient’sserum IC. ELISA’s records showed that over express FcγRⅡB can down regulate theantibody secretion capacity of Ramos cells.Conclusion Successfully prepared FcγRⅡB inducible expression lentiviral. Ramoscells which infected by this lentiviral can over express FcγRⅡB and over expression FcγRⅡBcan down regulate the antibody secretion capacity of Ramos cells.