Establishing transgene mouse models:construction and identification of human leucocyte antigen-A*0206 gene lentiviral vector / 中国组织工程研究

ABSTRACT

BACKGROUND:Studies have shown that human leukocyte antigen (HLA)-A*0206 subtype is related to the abscess of nasopharyngeal carcinoma, but there is no corresponding transgenic animal models that could used to judge the relationship between HLA-A*0206 and nasopharyngeal carcinoma on the overal level and further research of immunotherapy and gene therapy. OBJECTIVE:To construct lentiviral vectors carrying pLVX-CMV-HLA-A*0206-HA-mCMV-ZsGreen and establish HLA-A*0206 transgenic mice. METHODS:The HLA-A*0206 sequence was synthesized. EcoRI recognition site was introduced in the 5’ end by polymerase chain reaction, and influenza virus hemagglutinin labels and BamHI recognition site were introduced in the 3’ end. Eco RI and Bam HI double enzyme digestion target fragments and the pLVX-CMV-mCMV-ZsGreen plasmids were connected to the digested productions and transfected JM109 competent cel s. The positive clones were selected and identified by double enzyme digestion and sequencing. The positive plasmid and packaging plasmids were transfected into 293T cel s, which were human renal epithelial cel line that can express SV40 large T antigen. The lentivirus containing target sequence was produced. RESULTS AND CONCLUSION:Gel electrophoresis and sequencing results showed that, HLA-A*0206 was successful y inserted into pLVX-CMV-mCMV-ZsGreen frame plasmids. Transfection efficiency was 92%after 48 hours of transfecting 293T cel s. The viral suspension titer was 5 × 108 measured by fluorescence method. Experimental findings indicate that, the lentivirus containing cytomegalovirus promoter, HLA-A*0206, influenza virus hemagglutinin label and Zsgreen report gene was successful y constructed.