摘要
目的克隆宫颈癌病理组织中人乳头瘤病毒16型(HPV16)晚期蛋白(L1)的基因,并构建真核表达质粒,为制备预防性疫苗奠定基础。方法采用PCR法从宫颈癌病理标本中扩增出HPV16-L1基因,与pMD18T载体连接,测序并构建真核表达质粒(pVAX1L1),瞬时转染Cos7细胞,利用免疫组化技术检测表达产物。通过小鼠淋巴细胞转化试验检测免疫原性。结果所得L1基因的序列与GenBank中序列比较存在若干变异,并由此引起相应的氨基酸发生变化。pVAX1-L1瞬时转染Cos7细胞,经免疫组化技术检测到棕黄色免疫复合物的形成,说明有表达产物L1蛋白产生。淋巴细胞转化试验结果显示,试验组与对照组差异有显著意义。结论宫颈癌病理组织中HPV16L1基因序列存在一定程度的变异,所得L1基因表达产物具有生物学活性。
Objective To clone the gene encoding late protein L1 of human papillomavirus 16 （HPV16） from pathological tissue of cervical cancer, construct an eukaryotic expression vector and lay a foundation of preparation of vaccine for prophylactic use. Methods Amplify HPV16 L1 gene from the pathological tissue of cervical cancer and insert into vector pMD18-T. Identify the recom- binant plasmid by sequencing, then digest with restriction endonuclease and ligate to vector pVAX1. Transiently transfect Cos-7 cells with the constructed eukaryotic expression plasmid pVAX1-L1 and identify the expressed product by inmmunhistochemical technique. Immunize mice with the constructed recombinant plasmid pVAX1-L1 and determine the immunogenicity by lymphocyte transformation test,using plasmid pVAX1 as control. Results The amplified L1 gene sequence showed several variations as compared with that reported in GenBank,which caused the corresponding changes of amino acids. Brownish-yellow immune complex was detected in the transletted Cos-7 cells by immunohistochemical technique,which indicated the expression of L1 protein. The stimulating index of lymphocyte of mice immunized with plasmid pVAX1-L1 was signiflcandy higher than that with plasmid pVAX1. Conclusion The HPV16 L1 gene sequence amplified from cervical cancer tissue showed a certain variation. However,the expressed L1 protein show biological activity.
出处
《中国生物制品学杂志》
CAS
CSCD
2006年第3期259-261,共3页
Chinese Journal of Biologicals
