摘要
目的克隆弓形虫RH株Calpain(依钙蛋白酶)-like基因,分析免疫生物学功能,筛选预防弓形虫感染的疫苗候选分子。方法利用人类、鼠类和其它寄生虫的Calpain基因同源性比较的保守区域合成混合引物,以弓形虫的RNA为模板,应用RT-PCR扩增弓形虫Calpain-like基因片段,扩增的片段通过TA克隆插入克隆载体pET32A,筛选阳性克隆进行双酶切鉴定和DNA序列分析,用克隆的基因片段制备特异性基因探针,经Northern blot技术证实弓形虫Calpain-like基因mRNA。结果RT-PCR扩增了316bp弓形虫Calpain-like基因,其DNA序列与日本血吸虫Calpain基因同一区域的同源性为70%,与计算机推测的弓形虫Calpain-like基因的同源性为100%。Northern blot显示了弓形虫Calpain-like基因mRNA的大小约6300bp。结论弓形虫RH株基因组中存在Calpain-like基因,Calpain-like基因在弓形虫RH株中高度表达。
Objectlve To investigate molecular characteristics and biological properties of calpain - like gene from Toxoplasma gondii RH strain for future screening of calpaln vaccine candidates and target of drug prophylaxisby molecular clo- ning of this enzyme from Toxoplasma gondii RH strain, Methods RT - PCR and TA cloning technique were used to clone the fragment of calpain - like gene and then prokaryotic expression vector pET32A containing the fragment of calpain - like cD- NA was constructed by recombination techniques. Then specific genetic probe was prepared with the genetic fragment rooectly screened after enzymatic digestion and sequence analysis, Calpain - like mRNA was confirmed by Northern blotting. Results Calpain - like 316bp gene was cloned successfully by RT - PCR, The homology of of nucleotide sequence and amino acid sequence of calpain - like 316bp gene with Schistosomajaponicun was 70% and 100% with Toxoplasma gondii, respectively. Expression of calpain - like mRNA was investigated with a oligonucleotide probe corresponding to nucleotide positions of 6300bp. Conclusion The calpain - llke gene has been successfully expresssed in Toxoplasma gondil RH strain.
出处
《中国热带医学》
CAS
2008年第4期527-529,共3页
China Tropical Medicine
基金
广东省自然科学基金(No2003034971)
