微小隐孢子虫LDH基因的克隆与序列分析  

Cloning and sequencing analysis of LDH gene of Cryptosporidium parvum

作　　者：郭志云[1] 杨光[1] 荆春霞[1] 付芹芹[1] 孙小会[1] 王穗湘[1] 李月琴[2] 余新炳[3] 周天鸿[2] 

机构地区：[1]暨南大学医学院,广州510632 [2]暨南大学生命科学技术学院,广州510632 [3]中山大学基础医学院,广州510089

出　　处：《中国人兽共患病学报》2010年第12期1129-1133,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30901249);广东省自然科学基金(10151063201000036);广东省医学科研基金(B2007097)联合资助

摘　　要：目的克隆微小隐孢子虫(Cryptosporidiumparvum,Cp)南京株(NJ)乳酸脱氢酶(lactate dehydrogenase,LDH)基因,测序并分析微小隐孢子虫NJ株CpLDH与其他隐孢子虫分离株LDH基因序列的差异。方法根据微小隐孢子虫已知LDH基因序列设计合成2对引物,应用巢式PCR技术从微小隐孢子虫NJ株基因组DNA中扩增LDH基因,并将其克隆到pMD18-T载体上,阳性克隆的重组质粒经PCR及双酶切鉴定后,用双脱氧链终止法对重组质粒中的插入序列进行测序,应用生物信息学方法分析CpLDH基因序列和其他物种LDH序列的同源性。结果巢式PCR扩增得到特异的CpLDH基因序列,经PCR及双酶切鉴定获得了正确的pMD18-T-CpLDH重组质粒。测序表明,NJ株微小隐孢子虫LDH基因全长966bp,编码322个氨基酸,该基因序列已登录GenBank,登录号为HM001298。序列分析表明,我国微小隐孢子虫NJ株与国外分离的Iowa II株LDH基因编码的氨基酸序列具有98%的同源性。结论成功克隆了微小隐孢子虫NJ株LDH基因;序列测定及同源性分析表明,微小隐孢子虫NJ株在LDH酶关键结构位点存在突变。The propose of this study is to clone and determine the nucleotide sequences of LDH of Cryptosporidium parvum NJ strain,and analyze the sequences＇ differences between the NJ strain and others in the worldwide.Based on the given gene sequences of LDH,two pairs of primer were designed to amplify the LDH genes from the C.parvum NJ strain by Nested PCR technique,and was cloned into the pMD18-T vectors.Then the cloned gene from the positive recombinant plasmids were sequenced by dideoxy chain termination method after the identification of PCR and double digests methods.Last,bioinformatics methods were used to find out the homologies of the LDH gene between the C.parvum NJ strain and the other species.The CpLDH gene was specifically amplified by Nested PCR,and the correct recombinant plasmids were constructed after the identification by PCR and double digest methods.The results of sequencing of the nucleotides showed that the LDH gene of the C.parvum NJ strain was 966 bp in full length encoding 322 amino acids,and this gene sequence was logined to GenBank with the accession number of HM001298.And the sequence analysis showed that it had 98% homology in amino acids with the Iowa II strain abroad.The LDH gene of C.parvum NJ strain was successfully cloned,the results of sequencing and homology analysis indicate that there are mutations in the critical structure of CpLDH enzyme of C.parvum NJ strain.

关 键 词：微小隐孢子虫 乳酸脱氢酶 巢式PCR 生物信息学 

分 类 号：R382.3[医药卫生—医学寄生虫学]