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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。
Objective To clone the lactate dehydrogenase (LDH) gene of Cryptosporidium pulvum (Cp) Nanjing strain (NJ), and to sequence and analyze the difference of LDH gene sequence between CpLDH of Cryptosporidium parvum NJ and other Cryptosporidium isolates . Methods Based on the known LDH gene sequences of Cryptosporidium parvum, two pairs of primers were designed and synthesized. The nested PCR was used to amplify the LDH gene from the genomic DNA of Cryptosporidium parvum NJ and cloned into pMD18-T vector. The positive clones The recombinant plasmids were identified by PCR and restriction enzyme digestion. The inserted sequences in the recombinant plasmids were sequenced by dideoxy chain termination method and the homology of CpLDH gene sequences and LDH sequences of other species was analyzed by bioinformatics method. Results The specific CpLDH gene sequence was amplified by nested PCR. The correct pMD18-T-CpLDH recombinant plasmid was obtained by PCR and double enzyme digestion. Sequencing showed that the NJ strain Cryptosporidium parvum LDH gene was 966 bp in length and encoded 322 amino acids. The gene sequence was registered in GenBank with accession number HM001298. Sequence analysis showed that the amino acid sequence encoded by the NJH strain of Cryptosporidium parvum in China and the LDH gene of the Iowa II isolate in China were 98% homologous. Conclusion The LDH gene of Cryptosporidium parvum NJ was successfully cloned. Sequence analysis and homology analysis showed that there was a mutation in Cryptosporidium parvum NJ at the key structural site of LDH.