Construction and identification of a recombinant baculovirus transfer vector of conserved dopamine neurotrophic factor
10.3760/cma.j.issn.1671-8925.2010.08.005
- VernacularTitle: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
- Author:
Jun ZHANG
1
;
Chao-Shi NIU
;
Ge GAO
;
Jia-Ming MEI
;
Shen-Feng TANG
Author Information
1. 安徽医科大学附属省立医院
- Keywords:
Conserved dopamine neurotrophic factor;
Baculovirus transfer vector;
Parkinson's disease
- From:
Chinese Journal of Neuromedicine
2010;09(8):772-776
- CountryChina
- Language:Chinese
-
Abstract:
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.
