鼠B7-H4基因重组腺病毒的构建及其在B16F10细胞中的表达、鉴定

Construction of mB7-H4 recombinant adenovirus and its expression, identafication in B16F10

目的构建表达鼠B7-H4的重组腺病毒并检测其在B16F10细胞中的表达。方法采用RT-PCR技术,取小鼠肺脏,TriPure提取肺脏总RNA,反转录得到cDNA序列,PCR扩增B7-H4全长,克隆到plenti6/V5克隆载体中并经过测序证实与标准序列一致。加入适当的酶切位点,双酶切PCR扩增产物连接入pAd track CMV中构建成腺病毒穿梭质粒pAd track CMVmB7-H4,经酶切线性化后,用CaCl2的方法转化到含有腺病毒骨架质粒pAd Easy的BJ 5183大肠杆菌中,挑选同源重组菌落提取质粒,酶切线性化重组质粒并转染293细胞,包装成重组病毒颗粒。重组病毒上清感染B16F10细胞,并用PE标记的B7-H4单克隆抗体检测B16F10细胞mB7-H4蛋白的表达。结果RT-PCR扩增得到含编码mB7-H4的860 bp基因片段,与预期大小一致,并经过测序证实与标准序列一致。成功构建了mB7-H4重组腺病毒,病毒滴度达到3×1013pfu/ml,pAd mB7-H4重组腺病毒表达载体感染B16F10细胞后,荧光显微镜观察显示PE标记的mB7-H4单克隆抗体染色B16F10细胞阳性。结论成功构建了mB7-H4重组腺病毒,转染B16F10细胞后表达出有活性的B7-H4蛋白,为进一步研究B7-H4分子在器官移植、自身免疫性疾病以及肿瘤逃逸机制中的作用奠定了基础。

Objective To construct expression of mouse B7-H4 recombinant adenoviruses, and to identify the expression of mBT-H4 in B16F10 cell. Methods Mouse lung tissue was taken and its total RNA was extracted. Using RT-PCR, cDNA was reversed and mouse BT-H4 encoding sequence was amplified , gel purified,subcloned into plenti6/V5 vector ,verified by sequencing and put into pad track CMV vector in frame. The right plasmid was linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coil BJ5183 ceils with an adenoviral backbone plasmid, e.g. pad Easy. Recombinants are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease Pac I analyses. Finally, the Pac Ⅰ linearized recombinant plasmid was transfected into adenovirus packaging 293 cells to generated recombinant adenoviruses. Recombinant adenoviruses were transfected into B16F10 and the expressed mouse BT-H4 was identified by the PE labeled specified monoclonal antibody. Results The full-length of mBT-H4 about 860 bp , according to the expected value, was cloned from mouse lung tissure cDNA and was inserted into the Ad Easy expression plasmid. Recombinant adenoviruses was successfully generated with high titre to 3 ×10^13. The expression of mB7-H4 on B16F10 transfected with recombinant adenoviruses pAd mB7-H4 was verified by the fluorescence microscope and the expression of BT-H4 on B16F10 was clearly detected. Conclusion The recombinant adenoviruses pAdTmB7 H4 was successfully generated and the functional mBT-H4 expression on the B16F10 transfected with pAd-mB7-H4 was detected which firmly based the further study on the organ transplatation,autoimmune disease and mechanism of immune evasion in cancer research.