摘要
目的通过聚合酶链方法,以特异引物,从感染人乳头瘤病毒的新鲜食管癌组织获得人乳头瘤病毒16型晚期表达基因L_2保守区线性抗原片段的基因,并体外表达。获得具有抗原活性的原核表达蛋白,以探索研制低廉,方便及准确的人乳头瘤病毒感染的新型诊断试剂。方法在前人研究的基础上,经计算机检索,选定与食管癌高度相关的人乳头瘤病毒16型晚期表达基因L_2N端的一段基因,采用特异引物及聚合酶链方法从多例人乳头瘤病毒感染的食管癌组织DNA模板中,获得人乳头瘤病毒16型晚期表达基因L_2保守区线性抗原片段的基因,经克隆入测序载体验征DNA大小及序列后,将HPV16 L_2 DNA保守区线性抗原基因片段克隆入高效原核表达载体,并在大肠杆菌中表达,初步以Western-blot测定表达产物的抗原性:与乳头瘤病毒抗体及感染者血清的反应活性。结果以我们设计的特异引物,从两例人乳头瘤病毒感染阳性的食管癌组织DNA模板中能得到预期大小的PCR产物,并能插入预定的克隆载体中测序,所得基因的序列符合目的基因的序列,且两例组织所得的目的DNA序列完全一致,其插入大肠杆菌表达载体能获得表达,且表达产物具有抗原活性,能与商品化的乳头瘤病毒抗体及乳头瘤病毒感染患者的血清发生抗原抗体反应。结论从食管癌组织中能检出人乳头瘤病毒HPV16型晚期表达基因区L_2特定片段的基因,该片段确具高度保守性。在大肠杆菌中表达此片段所获得的表达产物,有抗原活性,有望用于流行病学检查,预警人乳头瘤病毒感染相关的癌症发生。
AIM To acquire the HPV16 L_2 conservative antigen DNA from tissues of esophageal carcinoma infected with human papillomavirus by PCR using special primers, and to sequence the subjective DNA by cloning into vector of pGEM-3zf(-) and express L_2 conservative antigen DNA in E. coli, and assay its immunoreaction with polyclone antibody or patients sera. METHODS The conservative antigen DNA segment was ascertained by comparing the L_2 sequence of human papillomavirus 16 through BLAST 2.0 on NCBI, then using specific primers, the subjective DNA segment was acquired by PCR from tissues of esophageal carcinoma infected with human papillomavirus. After that it was cloned into vector of pGEM-3zf(-) and sequenced, and inserted into an expression vector and expressed in E. coli. The antigen activity of expressed product was detected by Western-blot. RESULTS The desired DNA of HPV16 L_2 conservative antigen can be acquired by PCR using special primer from the tissues of esophageal carcinoma infected with human papillomaviruso the acquired DNA fragment can be cloned into vector of pGEM-3zf (-) and sequenced. The DNA sequence from two cases was highly conserved. It can also be cloned into an expression vector and expressed actively in E. coli. CONCLUSION The HPV16 L_2 conservative antigen DNA can be acquired from tissue of esophageal carcinoma Infected with human papillomavirus by PCR, and there is high conservation in the tissue of esophageal carcinoma infected with human papillomavirus. The product expressed in E. coil was active, which may provide a cheap and convenient method for epidemiology and presentiment of the occurence of cancer related with human papillomavirus.
出处
《世界华人消化杂志》
CAS
2001年第3期273-278,共6页
World Chinese Journal of Digestology
关键词
食管肿瘤
病毒学
乳头状瘤病毒
遗传学
基因病毒
基因表达
esophageal neoplasms/virology
papillomavirus, human/genetics
genes, viral
gene expression
