应用酵母双杂交技术筛选与pGBKT7-CT813编码产物相互作用蛋白的研究

Use of a yeast two-hybrid assay to identify proteins interacting with products expressed by pGBKT7-CT813

目的以pGBKT7-CT813作为诱饵质粒与HeLa细胞酵母GAL4AD融合cDNA文库进行酵母双杂交试验,以进一步探讨沙眼衣原体包涵体膜蛋白的生物学功能。方法根据STD基因库提供信息设计引物,用PCR法获取目的基因片段CT813,经酶切处理的CT813和pGBKT7质粒,在T4连接酶作用下连接,连接产物转入感受态细胞BL-21,培养后进行菌落PCR验证,对阳性质粒进行测序分析。质粒转化入酵母菌株Y187和AH109中,检测其有无自激活及毒性作用。阳性重组酵母菌株AH109与cDNA文库菌株Y187进行配合,待三叶草(或米奇)形状合子形成后涂布于腺嘌呤、组氨酸、亮氨酸、色氨酸缺陷型培养基和铺有X-Gal的SD/-Ade/-His/-Leu/-Trp平板上初筛,再经过2次筛选,收集阳性菌落。将阳性菌液点种在滤纸上,在液氮中反复冻融2次,然后浸泡在Z缓冲液-β巯基乙醇-X-Gal混合液中室温温育8h。对筛选出的显色阳性菌液进行PCR验证。选取22个PCR阳性菌液提取酵母质粒,将22个质粒再转入感受态细胞E.coli,BL-21,对阳性菌落提取质粒,进行回交试验,对验证阳性的菌落对应的质粒进行测序。结果成功构建了pGBKT7-CT813诱饵质粒,该质粒的表达产物对AH109和Y187均无自激活和毒性作用。将回交试验筛选的阳性菌落对应的质粒进行测序,对测序结果做BLAST检索分析,确定筛选出与pGBKT7-CT813特异性相互作用的基因可能编码的蛋白是:半乳糖凝集素-1(LGALS1)、环腺苷酸应答原件结合蛋白3(CREB3)、核糖体核糖体蛋白L10a(RPL10a)和微管蛋白37E-16(RP1-37E16)。结论筛选出的4种蛋白可能与沙眼衣原体的致病机制相关,但仍需要进一步验证。pGBKT7-CT813编码产物与多种蛋白有相互作用,为进一步研究其生物学功能打下了基础。

Objective A yeast two-hybrid assay was performed with the HeLa cDNA library and the bait vector pGBKT7-CT813for further in-depth study of the biological function of the inclusion membrane proteins of Chlamydia trachomatis. Methods Sequencing primers were designed to clone the target gene（CT813）according to the STDGEN database.The target gene was amplified using PCR.PCR products and the pGBKT7plasmid were digested with enzymes and then linked with T4ligase.The recombinants were transformed into competent cells（BL-21）that were cultured and then verified using colony PCR.The positive plasmid was sequenced.These recombinant plasmids were transformed into AH109and Y187yeast cells.The plasmids were identified,and neither had autoactivation or toxicity to yeast cells.The positive recombinant yeast strain AH109was combined with Y187from a cDNA library.Once clover-shaped or Mickey Mouse-shaped zygotes appeared,the mixture was coated on SD/-Ade/-His/-Leu/-Trp medium supplemented with X-Gal for preliminary screening.Positive colonies were collected after two rounds of screening.Drops of positive bacterial solution on filter paper were frozen in liquid nitrogen and then thawed in two cycles.Specimens were immersed in a mixture containing Z buffer,βmercaptoethanol,and X-Gal for eight hours.Positive bacterial solutions that were colored were verified using PCR.Twenty-two positive bacterial solutions were selected and the yeast plasmid that had been transformed into competent cells（BL-21）was extracted.Corresponding plasmids from positive bacteria colonies were sequenced. Results pGBKT7-CT813was successfully constructed as a bait plasmid and its expression products had no autoactivation and they were not toxic to AH109and Y187.Corresponding plasmids from positive bacteria colonies were sequenced aspart of retesting.Sequencing results were used in a BLAST search and analyzed.Proteins that were found to specifically interact with pGBKT7-CT813included Galectin-1（LGALS1）,cAMP response element binding protein 3（CREB3）,ribosomal protein L10a（RPL10a）,and tubulin 37E-16（RP1-37E16）. Conclusion Results indicated that these four proteins may be related to the pathogenic mechanism of C.trachomatis,but further verification is needed.Evidence of interaction between pGBKT7-CT813and various proteins has laid the foundation for further study of its biological function.