pCR3.1-bvLDH-C′_4的构建及在体内外的表达  

Construction of pCR3.1-bvLDH-C′_4 and Its Expression in Vivo and in Vitro

作　　者：常建军[1] 杨颖[1] 彭景楩[1] 

机构地区：[1]中国科学院动物研究所,计划生育生殖生物学国家重点实验室,北京100080

出　　处：《动物学杂志》2003年第1期28-33,共6页Chinese Journal of Zoology

基　　金：国家自然科学基金资助项目 (No .39870 10 0 )

摘　　要：精子特异性乳酸脱氢酶 (LDH C4)在精子的运动和存活等生理活动中起着重要的作用。研究表明 ,LDH C4接种鼠、兔等能够降低动物的生育率。通过RT PCR方法从布氏田鼠睾丸总RNA中克隆出编码抗原决定簇的LDH C4基因片断 ;采用T A克隆的方法将其插入到pCR3 1载体中构建重组载体pCR3 1 bvLDH C′4,测序结果表明克隆的基因片断与已知小鼠相应片断有 83 %的序列同源。通过质脂体法转染HeLa细胞 ,RT PCR证实其可在mRNA水平有效表达 ;通过肌肉接种BALB c小鼠 ,RTSperm specific lactate dehydrogenase(LDH C 4) is important in providing energy for the motility and survival of spermatozoa.There is abundant evidence in the literature that immunization with purified LDH C 4 reduces the fertibility of mice,rabbits and baboons.To study the immunological infertility of a recombinant vector of the wild mouse LDH C 4 gene, the cDNA sequence coding for the LDH C 4 fragment containing the antigen epitope C 5 15 was amplified from total testis RNA of Microtus branditi radde by reverse transcription and polymerse chain reaction (RT PCR).Then the cDNA fragment was inserted into the plasmid pCR3 1 to construct the recombinant vector pCR3 1 bvLDH C′ 4 by T A cloning.Sequencing revealed 83% homology with the known mouse LDH C 4 gene.Furthmore,when the recombinant plasmid was transfected into HeLa cells with lipofectamine,RT PCR sugests that these could efficiently express the mRNA of bvLDH C′ 4.BALB/c mice intramuscularly injected with the recombinant plasmid also expressed the mRNA of LDH C′ 4 at a relatively efficient level.

关 键 词：LDH-C4 布氏田鼠 基因疫苗 质脂体转染法 精子特异性乳酸脱氢酶 基因表达 pCR3.1-bvLDH-C4′ 

分 类 号：Q784[生物学—分子生物学]