旋毛虫肌幼虫p43cDNA的克隆及鉴定

Cloning and Characterization of p43 cDNA from Trichinella spiralis

目的克隆旋毛虫肌幼虫p43cDNA并对其表达的融合蛋白酶活性进行鉴定与分析。方法用PCR技术从旋毛虫肌幼虫cDNA文库中扩增靶基因,克隆到pMD-18T载体,转化至大肠埃希菌NovaBlue,序列测定后克隆到原核表达载体pET28a并转入表达菌DE3中。经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后提取包涵体并复性,通过降解双链λDNA测定其融合蛋白脱氧核糖核酸酶II(DNase II)活性。结果成功克隆到p43 cDNA序列,该cDNA序列与美国发表的存在两个核苷酸的变异,分别为第210位的C变为T,第604位的A变为G。基于3名研究者从旋毛虫T.spiralis美国分离株获得的序列相同、6名国内研究者(含本课题组)从T.spiralis中国分离株获得的序列也相同,由此可以确定这两个核苷酸的变异为T.spiralis中国分离株特异性和特征性单核苷酸多态性(SNP)标记。p43重组蛋白能够降解双链λDNA,表明其有DNase II酶活性。结论成功克隆到p43cDNA,T.spiralis中国分离株的p43cDNA具有两个SNP标记,其表达的重组蛋白具有DNase II酶活性。

Objective To clone and characterize the p43 cDNA from muscle larvae cDNA library of Trichinella spiralis （Ts） Chinese isolate. Methods PCR technique was used to amplify the target cDNA from muscle larvae cDNA library. After cloned in pMD18T vector, it was transformed into E. coli NovaBlue. The positive clones were sequenced and the cDNA was cloned into pET28a expression vector. After induced by IPTG, the inclusion body of the recombinant protein was purified and re-natured. The deoxyribonuclease Ⅱ （DNase Ⅱ ） activity of the recombinant protein was tested by hydrolyzing λDNA. Results Open reading frame （ORF） of the p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Chinese Ts, there were mutations of two nucleotides in the ORF of the Chinese Ts p43 cDNA comparing with that from USA isolate at the positions 210 and 604, namely, C and A in the USA isolate but T and G in the Chinese isolate. Considering that three authors had cloned the same p43 cDNA from the USA isolate and six groups （including this team） had also obtained the same sequence from the Chinese isolate, the mutation of the two nucleotides was considered as the single nucleotide polymorphic （SNP） marker of the Chinese Ts isolate. The DNase Ⅱ activity of the recombinant protein was successfully detected by hydrolyzing λDNA. Conclusion The p43 cDNA was successfully cloned from the muscle larvae cDNA library of the Ts Chinese isolate. Two SNPs were found in the nucleotide sequence. The DNase Ⅱ activity was proved.