γ1neo-hgp100基因的体外转染表达及体内免疫保护效应

The Expression and Protective Efficacy of DNA Vaccine Encoding Antibodized hgp100

目的:构建含有黑色素肿瘤相关抗原hgp100的CTL表位编码基因质粒γ1neo-hgp100,观察此质粒的体外表达和表达产物的提呈,以及体内基因免疫对黑色素肿瘤攻击的保护效应。方法:将编码hgp100的CTL表位的DNA序列插入抗体化抗原的表达质粒γ1neo中,构建γ1neo-hgp100,体外转染J558L,ELISA检测免疫球蛋白的表达;转染后的J558L与pmelTCR转基因T细胞共育,48h后ELISA检测培养上清中IFN-γ的含量。以γ1neo-hgp100脾内基因免疫C57BL/6小鼠,以B16F10黑色素瘤细胞攻击免疫后小鼠,定期观察肿瘤的生长情况及大小。结果:所构建的γ1neo-hgp100可以在体外表达,表达产物可以被充分的提呈,被提呈的hgp100CTL表位能有效的活化pmelTCRT细胞分泌IFN-γ。经γ1neo-hgp100免疫的小鼠能显著抵抗B16F10黑色素瘤细胞的攻击,生存时间得以明显延长。结论:γ1neo—hgp100基因可有效的在体外表达并被提呈至T细胞,产生体内免疫保护效应。

Objective: To investigate whether the plasmid γ1neo-hgp100 could be expressed and presented in vitro and could protect the immunized mice from B16F10 challenge in vivo. Methods: γ1neo-hgp100 plasmid was constructed in which the DNA sequence encoding hgp100 CTL epitope inserted into CDR3 of γ1-neo vector. The expression of anti-bodized antigen and IFN-γ in supernatant was measured by ELISA respectively after transfection J558L with γ1neo-hgp100 and further co-culture of J588L transfacted with γ1 neo-hgp100 and pmel TCR transgenic T cell. After introspleenic inoculation of γ1neo-hgp100, the protective efficacy of the gene vaccine was observed by means of measuring the tumor area every two days. Results: γ1neo-hgp100 could be expressed and presented in vitro, the immunogenecity of CTL epitope of hgp100 was strong enough and could activate gp100 specific T cell, the mice immunized with the gene vaccine could resist the tumor challenging in vivo. The mean survival time was prolonged to 36 days, compared to control group (P < 0. 05 ). Conclusion: γ1neo-hgp100 could be expressed and presented in vitro and protect mice from tumor challenging.