保守性多巴胺能神经营养因子重组杆状病毒转移载体的构建及鉴定

Construction and identification of a recombinant baculovirus transfer vector of conserved dopamine neurotrophic factor

目的 构建并鉴定小鼠保守性多巴胺能神经营养因子（mCDNF）重组杆状病毒转移载体pFastBacHTb-mCDNF. 方法 应用Trizol法提取小鼠组织总RNA,反转成cDNA,经PCR扩增得到带有预定酶切位点（BamH Ⅰ、Xho Ⅰ）的mCDNF基因全长（564 bp）,回收片段并克隆至pGEM-T载体,测序验证PCR结果的准确性.将mCDNF定向克隆到pFastBacHTb载体,构建含有mCDNF基因的重组质粒pFastBacHTb-mCDNF,转化大肠杆菌DH5α感受态细胞,氨苄青霉素抗性筛选阳性克隆,摇菌抽取质粒进行测序和双酶切鉴定. 结果 RT-PCR扩增产物经琼脂糖凝胶电泳显示得到预定大小的目的 条带（564 bp）,mCDNF的T-A克隆经蓝白斑抗性筛选获得阳性克隆,PCR及测序均提示pGEM-T-CDNF载体成功构建.重组质粒pGEM-T-mCDNF和pFastBacHTb载体进行BamH Ⅰ、Xho Ⅰ限制性内切酶酶切后再连接,得到pFastBacHTb-mCDNF重组质粒,并经PCR、酶切及测序验证无误. 结论 本实验成功构建了mCDNF重组杆状病毒转移载体pFastBacHTb-mCDNF,为该营养因子的进一步研究奠定了一定基础.

Objective To construct and identify a recombinant baculovirus transfer vector of mouse conserved dopamine neurotrophic factor （mCDNF）： pFastBacHTb-mCDNF. Methods Mouse total RNA was isolated by using Trizol reagent, and then, first-strand cDNAs were synthesized by reverse transcriptase. Overall length of CDNF （564 bp） was amplified by two rounds of PCR introducing appropriate restriction sites （BamH Ⅰ, Xho Ⅰ）. The PCR products were cloned into pGEM-T vector and sequenced to confirm PCR fidelity. The mCDNF was sub-cloned into pFastBacHTb vector to create pFastBacHTb-mCDNF vector, then the vector was transferred into the E. coli DH5α competent cells. The clone was selected using amicillin resistance and then this vector was sequenced and identified by double digests. Results Agarose gel electrophoresis after RT-PCR showed a 564 bp band being consistent with the anticipation size. Positive clone of pGEM-T-CDNF was screened by blue/white and antibiotic resistance selection. Recombinant plasmid pGEM-T-mCDNF was identified by PCR and sequence.Recombinant plsmid pGEM-T-mCDNF and pFastBacHTb vector were cut by BamH Ⅰ and XhoⅠ restriction enzyme, and then, recombinant plasmid pFastBacHTb-mCDNF was constructed and successfully identified by double digestion of Xho Ⅰ and BamH Ⅰ restriction enzyme or single digestion of BamH Ⅰ, PCR and sequence. Conclusion We successfully constructe the recombinant baculovirus transfer vector pFastBacHTb-mCDNF, laying the foundation for further research of this neurotrophic factor.