BDNF基因逆转录表达载体的构建和在淋巴细胞中的表达

【目的】构建含脑源性神经营养因子基因(brain-derivedneurotrophicfactor,BDNFgene)逆转录表达载体并了解其在淋巴细胞中的表达,探讨治疗基因的非损伤性脑内转移的新途径。【方法】用RT-PCR方法从大鼠海马组织总RNA中扩增(cDNA)BDNF片段,应用基因重组技术将大鼠(cDNA)BDNF克隆到逆转录病毒载体pLXSN中,用限制性内切酶酶切分析和DNA测序对重组质粒进行鉴定,采用LipofectAMINE将重组大鼠(cDNA)BDNF导入PA317细胞,获取高滴度的病毒上清转染大鼠淋巴细胞,应用流式细胞仪(FCM)检测大鼠淋巴细胞BDNF的表达,噻唑蓝(MTT)法检测淋巴细胞培养上清对PC12细胞增殖的影响。【结果】扩增出大鼠(cDNA)BDNF片段,限制性内切酶酶切和DNA测序证实获得正确的重组质粒pLXSN-BDNF,BDNF基因修饰的大鼠淋巴细胞能高表达BDNF蛋白,其培养上清能促进PC12细胞增殖。【结论】成功构建了BDNF基因逆转录表达载体,BDNF基因修饰的大鼠淋巴细胞能高表达和分泌具有生物活性的BDNF蛋白,为治疗基因的非损伤性脑内转移奠定了一定基础。

Construction of Recombinant Retroviral Vector Carrying Rat BDNF Gene and its Expression in Lymphocytes

Objective] To construct the recombinant retroviral vector carrying rat brain derived neurotrophic factor gene (BDNF gene)and to investigate its expression in lymphocytes. The cDNA encoding the rat BDNF was amplified by RT PCR and was inserted into polylinker site of retroviral vector pLXSN through genetic recombination technique. The recombinant plasmid was verified with restriction analysis and DNA sequencing. Recombined (cDNA) BDNF plasmid was transfected by LipofectAMINE into the packing cell line PA317 and G418 resistant clones with highest titer was selected. Rat lymphocytes were infected repeatedly with virus supernatant. The expression of BDNF protein in rat lymphocytes was assayed by FCM. The proliferation of PC12 cells was evaluated by MTT assay. The rat (cDNA) BDNF was amplified. The restriction analysis and DNA sequencing proved that the plasmid pLXSN BDNF was obtained. BDNF protein expressed in rat lymphocytes modified by BDNF gene. The supernatant of lymphocytes modified by BDNF gene enhanced the proliferation of PC12 cells. [ Conclusion] The plasmid pLXSN BDNF is successfully constructed. Rat lymphocytes modified by BDNF gene can express and secrete active BDNF protein in vitro and may be used as a tool of non traumatic transmission of therapeutic gene into central nervous system (CNS) tissue .