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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 p43 cDNA of Trichinella spiralis muscle larvae and identify and analyze the expression of fusion proteinase. Methods The target gene was amplified from the cDNA library of Trichinella spiralis larvae by PCR and cloned into pMD-18T vector. The target gene was transformed into Escherichia coli NovaBlue and cloned into prokaryotic expression vector pET28a and transformed into E. coli DE3. After IPTG induction, the inclusion body was extracted and renatured. The DNase II activity of the fusion protein was determined by degradation of double-stranded λDNA. Results The p43 cDNA sequence was successfully cloned. This cDNA sequence is identical to the published nucleotide sequence in the United States. The nucleotide sequence at position 210 is T and the A at position 604 is G. Based on the same sequence obtained from three S. Trichinella strains in the United States, three domestic researchers obtained the same sequence from the T.spiralis Chinese isolate from six domestic researchers (including our group) The nucleotide variation was a T.spiralis Chinese isolate-specific and characteristic single nucleotide polymorphism (SNP) marker. The p43 recombinant protein is capable of degrading double-stranded λDNA, indicating that it has DNase II enzyme activity. Conclusion The p43 cDNA was successfully cloned into p43 cDNA. The p43 cDNA of T.spiralisis isolates in China has two SNP markers, and the expressed recombinant protein has DNase II activity.