VP1Cm—一种对沙眼衣原体高效抑制蛋白的构建  

VP1Cm—Construction of a Highly Inhibitory Protein Against Chlamydia Trachomatis

作　　者：陈晗 赵乐然[1] 尤聪[1] 孔杰[1] 邵丽丽[1] 刘全忠[1] CHEN Han;ZHAO Leran;YOU Cong;KONG Jie;SHAO Lili;LIU Quanzhong(Department of Dermatology and Venereology,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院皮肤性病科,天津300052

出　　处：《中国皮肤性病学杂志》2020年第9期1012-1017,共6页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金(31870169)。

摘　　要：目的设计构建针对沙眼衣原体(Chlamydia trachomatis,Ct)具有高效抑制作用的重组蛋白,为衣原体治疗抵抗探索新的解决方法。方法通过生物信息学方法设计VP1Cm蛋白的基因序列。应用基因合成的方法构建重组质粒VP1Cm-pET28a(+)并转化至大肠杆菌中。用带Ni^+的亲和树脂纯化蛋白,梯度透析复性蛋白。CCK8法测定重组蛋白对Hela细胞的毒性作用。将豚鼠嗜衣原体噬菌体PhiCPG1衣壳蛋白VP1、截短蛋白VP1C、蛋白VP1Cm、杆菌肽和磷酸盐缓冲溶液(PBS)分别与E型Ct标准株于室温下预孵育3 h后接种至Hela细胞,同时设置Ct感染对照组。48 h后免疫荧光染色计数包涵体。多组样本均数间比较用单因素ANOVA分析,两样本均数间比较用Bonferroni检验。结果成功表达并纯化出蛋白VP1Cm。在相同浓度时,PhiCPG1衣壳蛋白VP1、截短蛋白VP1C和蛋白VP1Cm对Ct的抑制率分别为76.20%、85.13%和90.01%。结论经设计过的蛋白VP1Cm对Ct的抑制效果优于PhiCPG1衣壳蛋白VP1及其功能结构域截短蛋白VP1C。Objective To design and construct a recombinant protein with high inhibitory effect on Chlamydia trachomatis(Ct) and to explore a new solution for the treatment resistance of Chlamydia trachomatis.Methods The gene sequence of VP1 Cm protein was designed by bioinformatics method.The recombinant plasmid VP1 Cm-pET28 a(+) was constructed by gene synthesis and then transformed into E.coli.The protein was purified by affinity resin with Ni+ and renatured by gradient dialysis.The toxic effect of recombinant protein on Hela cells was determined by CCK8 method.The PhiCPG1 capsid protein VP1,truncated protein VP1 C,protein VP1 Cm,bacitracin and phosphate buffer solution(PBS) were respectively preincubated with E-type Ct standard strain at room temperature for 3 hours and then inoculated into Hela cells, Ct infection control group was set up at the same time,the inclusions were counted by immunofluorescence after 48 hours.Single factor ANOVA analysis was used to compare the mean of multiple samples,and Bonferroni test was used to compare the mean of the two samples.Results The protein VP1 Cm was successfully expressed and purified.At the same concentration,the inhibition rates of PhiCPG1 capsid protein VP1,truncated protein VP1 C and protein VP1 Cm on Ct were 76.20%,85.13% and 90.01%,respectively.Conclusion The designed protein VP1 Cm had a better inhibitory effect on Ct than PhiCPG1 capsid protein VP1 and its functional domain truncated protein VP1 C.

关 键 词：沙眼衣原体 衣原体噬菌体 PhiCPG1 VP1 

分 类 号：R518[医药卫生—内科学]