周期型马来丝虫GAPDH部分编码基因原核及真核表达载体的构建  

CONSTRUCTION OF PROKARYOTIC AND EUKARYOTIC EXPRESSION PLASMIDS WITH GAPDH GENE FROM PERIODIC BRUGIA MALAYI

作　　者：张赛楠[1] 方政[1] 童海燕[1] 方浩[1] 谢东方[1] 黄为群[1] 徐邦生[1] 

机构地区：[1]南通大学医学院寄生虫学教研室,南通226001

出　　处：《现代预防医学》2010年第22期4304-4306,4311,共4页Modern Preventive Medicine

基　　金：江苏省社会发展科技计划项目(BS2006522)

摘　　要：[目的]构建我国流行的周期型马来丝虫3-磷酸甘油醛脱氢酶(BmGAPDH)部分编码基因原核和真核表达质粒以及基因序列分析,为进一步的研究奠定基础。[方法]根据GeneBank中马来丝虫GAPDH基因的已知序列设计引物,以周期型马来丝虫总RNA为模板,反转录PCR(RT-PCR)扩增目的编码基因。扩增产物经初步鉴定后将其克隆入pGEM-T载体,转化大肠杆菌(E.coli)DH5α,筛选阳性克隆,进行双酶切及PCR扩增鉴定,获得阳性重组质粒pGEM-BmGAPDH,经测序验证,并进行同源性比较。亚克隆至真核表达质粒pcDNA3.1(+),构建真核表达载体pcD-NA3.1(+)-BmGAPDH,转染COS-7细胞后进行RT-PCR验证。[结果]RT-PCR扩增出一条约877 bp大小的特异性条带,重组质粒双酶切的PCR结果与预期相符,DNA序列分析与GeneBank已知的基因序列同源性为99%。转染的COS-7细胞高水平表达BmGAPDH的mRNA,根据克隆的目的基因序列推导的氨基酸序列与GenBank登录的一致。[结论]成功构建了周期型马来丝虫3-磷酸甘油醛脱氢酶部分编码基因原核及真核表达载体,为进一步功能研究提供了条件。[Objective] To construct the prokaryotic and eukarvotic expression plasmids containing the glyceraldehydes- 3-phosphate dehydrogenase （GAPDH） from periodic Brugia malayi so that to provide for basis of immunological prevention. [Methods] The total RNA was extracted from periodic Brugia malayi. A couple of specific primers were designed on the basis of known sequences of GAPDH gene from B. malayi. The desired gene was amplified by PCR technique from cDNA. The prod- ucts were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coil （E. coil） strain DH5ct. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid of pGEM-T-BmGAPDH was confirmed by sequencing and homology comparison. The PCR product was cloned and then subcloned into pcDNA3.1 （＋） vector. The recombinant eukaryotic plasmids was screened and identified by digestion with restriction enzyme and PCR amplification, and was transfected into COS-7 cell subsequently. [ Results] A spe- cific band of around 877 bp was amplified by RT-PCR. The same product was obtained by double restriction enzyme digestion of recombinant plasmid and PCR. The result of DNA sequencing showed that BmGAPDH shares 99% nucleotide sequence with that of known sequences in the gene bank. BmGAPDH mRNA was highly expressed in transfected COS-7 ceil. The deduced amino acid sequence showed to be identical with that of BmGAPDH. [ Conclusion ] The recombinant plasmid pcDNA3.] （＋） -Bm- GAPDH has been successfully constructed, which provides the basis for further study of BmGAPDH expression and functions.

关 键 词：周期型马来丝虫 3-磷酸甘油醛脱氢酶 真核表达载体 COS-7细胞 

分 类 号：R383.16[医药卫生—医学寄生虫学]