Packaging and identification of miR-137 overexpression lentivirus / 吉林大学学报(医学版)

Objective:To construct lentiviral vector which can overexpression miR-137 and produce lentivirus by lentivirus packaging system, and to explore its infection efficiency and expression in HEK293T cells.Methods: miR-137 sequence was chemically synthesized and cloned into lentiviral vector GV209, and the recombinant plasmid containing human miR-137 was obtained and identified.Then miR-137 recombinant plasmid together with two helper plasmids were transfected into HEK293T cells using Lipofectamine 2000.After the HEK293T cells were infected in multiplicity of infection(MOI) 40 for 48 h, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the expression level of miR-137 was detected by fluorescence quantitative PCR.Results:The sequencing results showed that the inserted gene sequence was completely consistent with the published human miR-137 gene sequence in GenBank.The GFP was observed in the HEK293T cells infected with miR-137 overexpression lentivirus under fluorescence microscope.The fluorescence quantitative PCR results showed that the expression level of miR-137 in the cells infected with overexpression lentivirus was 12.74 times higher than that in the control cells.Conclusion:The lentivirus containing miR-137 gene is successful packaged, and it could efficiently infect the HEK293T cells.

Preparation of two mouse anti-chicken CARP monoclonal antibodies and identification of their biological characteristics / 中国免疫学杂志

Objective:To prepare the monoclonal antibodies against chicken CARP.Methods:The chicken CARP cDNA encoding the N-terminal 110aa length of encoding region was cloned into prokaryotic expression vector pET28b.The BALB/c mice were immunized with purified the recombinant protein,spleen cells of mouse were fused with SP2/0,and the positive clones were screened by indirect ELISA.The properties of McAb were analyzed by ELISA and Western blot.Results:Two hybridomas were proved to have the capability of stably secreting McAb against chicken CARP,designated as 4A8 and 4E6,the subtype of both was IgG1,and the light chain was ?.The titers of ascites were 3.2?104 and 1.28?105 respectively.The Western blot results suggested that they could recognize chicken CARP protein.Conclusion:Two murine hybridomas secreting anti-chicken CARP McAbs have been successfully obtained.