摘要
目的:构建小鼠B7-H4(mB7-H4)慢病毒表达载体并观察B7-H4蛋白对脾细胞增殖的影响。方法:从BALB/c小鼠肺脏中克隆B7-H4基因,构建表达载体mB7-H4-EGFP,酶切后克隆入慢病毒表达载体pCDH1-MCS1-EF1-Puro中,命名为pCDH1-mB7-H4-EGFP;以包含起始密码和多克隆位点的寡核苷酸取代mB7-H4作为对照载体pCDH1-Control-EGFP。分别与包装载体混合物一起经LipofectamineTM2000转染入293T细胞包装成病毒颗粒,感染293T细胞。pCDH1-mB7-H4-EGFP重组慢病毒感染的293T细胞分别与BALB/c、C57BL/6、BALB/c∶C57BL/6(1∶1)小鼠脾细胞混合培养,用MTT法检测其对脾细胞增殖的影响,对照为pCDH1-Con-trol-EGFP重组慢病毒感染的293T细胞进行的相同处理。结果:mB7-H4被成功克隆,测序证实与GenBank的mRNA核酸序列2个核苷酸有差异,但与其编码的氨基酸一致。pCDH1-mB7-H4-EGFP慢病毒表达载体被成功构建。mB7-H4蛋白表达在293T细胞表面,与对照相比,对BALB/c、C57BL/6、BALB/c∶C57BL/6(1∶1)小鼠脾细胞增殖均具有明显抑制作用。结论:成功构建小鼠B7-H4慢病毒表达载体,感染293T细胞后表达出对脾细胞增殖具有抑制活性的mB7-H4膜表面蛋白。
Objective：To construct lentivirus expression vector of mouse B7-H4 and investigate its effect on the proliferation of mouse splenocytes.Methods：B7-H4 gene was cloned from normal lung tissue of BALB/c mouse.mB7-H4-EGFP vector was constructed.The mB7-H4-EGFP fusion gene was spliced out and ligated into lentivirus expression vector pCDH1-MCS1-EF1-puro.The resulting plasmid was designated pCDH1-mB7-H4-EGFP.The control vector pCDH1-Control-EGFP was constructed with a DNA oligonucleotide containing initiating Met and a multiple cloning site to replace mB7-H4.pCDH1-mB7-H4-EGFP or pCDH1-Control-EGFP was co-transfected into 293T cells with packaging plasmid mix by LipofectamineTM 2000 reagent.The supernatant of the cultured 293T cells was collected and used to infect 293T cells.MTT assay was used to assess the proliferation of BALB/c,C57BL/6 or BALB/c∶ C57BL/6（1∶ 1）mouse splenocyte that was co-cultured with mB7-H4 or control protein expressing 293T cells.Results：mB7-H4 was successfully cloned.Sequence analysis showed two nucleotides were different from those reported mB7-H4 sequence in GenBank,whereas their deduced amino acid sequences were consistent.mB7-H4 lentivirus expression vector were successfully constructed.mB7-H4-EGFP was expressed on the surface of infected 293T cells,and could inhibit the proliferation of BALB/c,C57BL/6 or BALB/c∶ C57BL/6（1∶ 1） mouse splenocyte.Conclusion：The mouse B7-H4 lentivirus expression vector was successfully constructed,and the functional mB7-H4 protein was detected on the surface of infected 293T cells.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第3期200-204,共5页
Chinese Journal of Immunology
基金
四川省科技厅应用基础课题(No.04JY029-014-2)
