人乳头瘤病毒16型E2基因的克隆、表达及鉴定
摘要
宫颈癌是妇科恶性肿瘤死亡的主要病因。分子生物学研究发现,高危型人乳头瘤病毒(human papillomavirus,HPV)感染与90%以上的宫颈癌有着密切的联系,进而发现尤其是HPV16型在各地区HPV亚型分布中居于主导地位。高危型人乳头瘤病毒以两种方式途径感染宫颈上皮细胞。在HPV急性感染中,完整病毒DNA游离地存在于宿主细胞,并自主完成生活周期,产生新的感染病毒颗粒。而在少数HPV感染病人身上,病毒环状基因组整合进宿主细胞染色体,导致高级宫颈内皮瘤病变,甚至可能发展为浸润性宫颈癌。感染病毒的早期基因E2丧失功能,E6持续表达在诱发宫颈癌的过程中发挥着重要作用。目前,高危型HPVE2与宫颈癌细胞E6表达和细胞增殖的关系,以及其表达抑制宫颈癌发生发展的机制仍是尚待解决的问题。为了探讨HPV16型E2基因对宫颈癌发展过程的影响,本研究在原核表达系统中诱导表达并纯化E2蛋白,制备抗HPV16E2抗血清。同时,在SiHa细胞中表达外源基因HPV16E2,以鉴定E2蛋白表达对HPV16E6基因表达和SiHa细胞生长状态的影响。
研究结果初步表明,E2蛋白能够在大肠杆菌BL21中表达,纯化蛋白在SDS-PAGE电泳上显示单一条带。ELISA和Western blotting检测表明获得的E2抗体具免疫特异性。半定量PCR和细胞计数结果可分别说明,HPV16E2在SiHa细胞中的表达能够抑制内源基因HPV16E6 mRNA表达,并显著降低SiHa细胞的增殖数。由上述实验结果推测,HPV16E2可以结合早期基因启动子,控制人乳头瘤病毒致癌基因的表达,抑制细胞无限增殖,从而对癌症治疗起到一定作用,这为进一步探索宫颈癌的基因治疗方案提供了线索。
研究主要包括以下几部分内容:
1. HPV16E2基因原核、真核表达载体的构建
根据人乳头瘤病毒16E2基因cDNA序列设计一对引物,采用PCR的方法从新疆维吾尔族妇女宫颈癌样本中扩增HPV16E2基因的目的片段并将其插入到原核表达载体pMAL-p2X中。再由构建好的pMAL/HPV16E2重组质粒亚克隆目的基因片段,纯化回收后插入真核表达载体pCDNA3中,构建HPV16 E2基因的真核表达载体pCDNA3/HPV16E2。
2. HPV16E2基因原核表达及抗血清的制备
为进一步研究HPV16E2基因的功能,将构建好的重组质粒pCDNA3/HPV16E2,免疫小鼠。原核表达重组质粒转化大肠杆菌BL21,加入IPTG诱导表达MBP/E2融合蛋白,表达产物用麦芽糖亲和层析柱纯化。将纯化后的重组蛋白作为检测抗原,ELISA和Western blotting结果表明pCDNA3/HPV16E2免疫小鼠产生了特异性强的抗体。本研究所构建的真核表达载体pCDNA3/HPV16E2及所获得的抗HPV16E2抗体,为在细胞水平上研究HPV16E2基因表达及基因功能提供了有力的工具。
3. HPV16E2表达对SiHa细胞生长的影响
利用脂质体转染法将真核表达质粒pCDNA3/HPV16E2导入SiHa细胞,表达E2蛋白,观察转染SiHa细胞中HPV16E6 mRNA表达及细胞增殖的情况。RT-PCR和Western blotting结果分别显示E2在SiHa细胞中产生mRNA和蛋白质水平的表达。与转染pCDNA3空载体的对照组比较,转染pCDNA3/HPV16E2组的HPV16E6 mRNA表达明显受到抑制,且SiHa细胞增殖数显著低于对照组(P<0.05)。
Cervical cancer is one of the leading causes of cancer-related death in women.Evidences from Human Papillomavirus indicated that more than 90% cervical cancer development was a complex process associated with high-risk HPV infection.HPV type 16 (HPV-16) was related to it predominate.High-risk HPV infection of the cervical epithelium occurs in two forms.In acute infections,a complete copy of the HPV viral DNA is present as an episome within the host cell,and the virus is capable of completing its life cycle,producing new,infectious viral particles.In a small minority of HPV-infected patients,the circular viral genome is integrated into the host-cell DNA, producing high-grade dysplasia,which can progress to invasive carcinoma.Among these genes, HPV16E2 and HPV16E6 gene play important roles in cervical cancer regulation.The correlation among E2 gene,E6 gene and cancer cell division,as well as the E2 gene function are still suspensive.In the current study,to discuss whether there is any effect produced by E2 on cervical cancer development, E2 gene was expressed in E.coli.and purified by affinity chromatography.then anti-HPV16E2 antibody was prepared.Meanwhile,the E2 recombinant plasmid was transfected into SiHa cells,then the effects on the HPV16E6 mRNA expression and the SiHa cell growth were analysed.
In this study,The SDS-PAGE results suggested that in Escherichia coli BL21, the E2 fusion protein was expressed and purified. The active immunization results showed that antibody against E2 was specific.The pCDNA3/HPV16E2 group had significant lower HPV16 E6 mRNA content and cell growth number than control group detected by Semi-Quantitative RT-PCR and cell counting.Therefore,HPV16E2,binding the early gene promoter and regulating HPVE6 gene expression, can restrain cell growth and division, which will provide the cue for cervical cancer therapy.
The research comprises as follows:
First, the construction of expression vector of HPV16E2 gene from HPV.
To construct the prokaryotic and eukaryotic expression vector and prepare the antiserum of HPV16E2 gene. The sequence of HPV16E2 gene was amplified by reverse transcriptase-PCR (RT-PCR) from the total RNA of cervical cancer cells and insert into pMAL vector and pCDNA3 vector.
Secondly,the HPV16E2 prokaryotic expression and preparation of HPV16E2 gene antiserum.
To further study the function of HPV16E2 gene,HPV16E2 were cloned into pCDNA3 to form pCDNA3/HPV16E2 DNA vaccines,and then vaccinate mice by DNA delivery way.Fusion protein MBP/E2 were expressed in E.coli.BL21,into which prokaryotic expression plasmids pMAL/HPV16E2 was introduced with IPTG induction. The expressed fusion proteins were purified by MBP affinity chromatography.ELISA and Western blotting showed that antibody obtained from the serum of mice by injecting recombinant plasmid pCDNA3/HPV16E2 were strong specific.The recombinant plasmid pCDNA3/HPV16E2 and anti-HPV16E2 antibody in the current study would provide a powerful tool for function research of HPV gene in cell level;
Finally,pCDNA3/HPV16E2 effects on SiHa cells growth number in detection.
pcDNA3/HPV16E2 was transfected into SiHa cells and E2 protein was expressed.The HPV16E6 mRNA expression and SiHa cell growth were detected.RT-PCR and Western blotting result respectively showed that in SiHa cells E2 mRNA and protein were expressed.In HPV16E2 gene transfection test, the pCDNA3/HPV16E2 group had significant lower HPV16E6 mRNA level and cell growth number than control group(P<0.05).
研究结果初步表明,E2蛋白能够在大肠杆菌BL21中表达,纯化蛋白在SDS-PAGE电泳上显示单一条带。ELISA和Western blotting检测表明获得的E2抗体具免疫特异性。半定量PCR和细胞计数结果可分别说明,HPV16E2在SiHa细胞中的表达能够抑制内源基因HPV16E6 mRNA表达,并显著降低SiHa细胞的增殖数。由上述实验结果推测,HPV16E2可以结合早期基因启动子,控制人乳头瘤病毒致癌基因的表达,抑制细胞无限增殖,从而对癌症治疗起到一定作用,这为进一步探索宫颈癌的基因治疗方案提供了线索。
研究主要包括以下几部分内容:
1. HPV16E2基因原核、真核表达载体的构建
根据人乳头瘤病毒16E2基因cDNA序列设计一对引物,采用PCR的方法从新疆维吾尔族妇女宫颈癌样本中扩增HPV16E2基因的目的片段并将其插入到原核表达载体pMAL-p2X中。再由构建好的pMAL/HPV16E2重组质粒亚克隆目的基因片段,纯化回收后插入真核表达载体pCDNA3中,构建HPV16 E2基因的真核表达载体pCDNA3/HPV16E2。
2. HPV16E2基因原核表达及抗血清的制备
为进一步研究HPV16E2基因的功能,将构建好的重组质粒pCDNA3/HPV16E2,免疫小鼠。原核表达重组质粒转化大肠杆菌BL21,加入IPTG诱导表达MBP/E2融合蛋白,表达产物用麦芽糖亲和层析柱纯化。将纯化后的重组蛋白作为检测抗原,ELISA和Western blotting结果表明pCDNA3/HPV16E2免疫小鼠产生了特异性强的抗体。本研究所构建的真核表达载体pCDNA3/HPV16E2及所获得的抗HPV16E2抗体,为在细胞水平上研究HPV16E2基因表达及基因功能提供了有力的工具。
3. HPV16E2表达对SiHa细胞生长的影响
利用脂质体转染法将真核表达质粒pCDNA3/HPV16E2导入SiHa细胞,表达E2蛋白,观察转染SiHa细胞中HPV16E6 mRNA表达及细胞增殖的情况。RT-PCR和Western blotting结果分别显示E2在SiHa细胞中产生mRNA和蛋白质水平的表达。与转染pCDNA3空载体的对照组比较,转染pCDNA3/HPV16E2组的HPV16E6 mRNA表达明显受到抑制,且SiHa细胞增殖数显著低于对照组(P<0.05)。
Cervical cancer is one of the leading causes of cancer-related death in women.Evidences from Human Papillomavirus indicated that more than 90% cervical cancer development was a complex process associated with high-risk HPV infection.HPV type 16 (HPV-16) was related to it predominate.High-risk HPV infection of the cervical epithelium occurs in two forms.In acute infections,a complete copy of the HPV viral DNA is present as an episome within the host cell,and the virus is capable of completing its life cycle,producing new,infectious viral particles.In a small minority of HPV-infected patients,the circular viral genome is integrated into the host-cell DNA, producing high-grade dysplasia,which can progress to invasive carcinoma.Among these genes, HPV16E2 and HPV16E6 gene play important roles in cervical cancer regulation.The correlation among E2 gene,E6 gene and cancer cell division,as well as the E2 gene function are still suspensive.In the current study,to discuss whether there is any effect produced by E2 on cervical cancer development, E2 gene was expressed in E.coli.and purified by affinity chromatography.then anti-HPV16E2 antibody was prepared.Meanwhile,the E2 recombinant plasmid was transfected into SiHa cells,then the effects on the HPV16E6 mRNA expression and the SiHa cell growth were analysed.
In this study,The SDS-PAGE results suggested that in Escherichia coli BL21, the E2 fusion protein was expressed and purified. The active immunization results showed that antibody against E2 was specific.The pCDNA3/HPV16E2 group had significant lower HPV16 E6 mRNA content and cell growth number than control group detected by Semi-Quantitative RT-PCR and cell counting.Therefore,HPV16E2,binding the early gene promoter and regulating HPVE6 gene expression, can restrain cell growth and division, which will provide the cue for cervical cancer therapy.
The research comprises as follows:
First, the construction of expression vector of HPV16E2 gene from HPV.
To construct the prokaryotic and eukaryotic expression vector and prepare the antiserum of HPV16E2 gene. The sequence of HPV16E2 gene was amplified by reverse transcriptase-PCR (RT-PCR) from the total RNA of cervical cancer cells and insert into pMAL vector and pCDNA3 vector.
Secondly,the HPV16E2 prokaryotic expression and preparation of HPV16E2 gene antiserum.
To further study the function of HPV16E2 gene,HPV16E2 were cloned into pCDNA3 to form pCDNA3/HPV16E2 DNA vaccines,and then vaccinate mice by DNA delivery way.Fusion protein MBP/E2 were expressed in E.coli.BL21,into which prokaryotic expression plasmids pMAL/HPV16E2 was introduced with IPTG induction. The expressed fusion proteins were purified by MBP affinity chromatography.ELISA and Western blotting showed that antibody obtained from the serum of mice by injecting recombinant plasmid pCDNA3/HPV16E2 were strong specific.The recombinant plasmid pCDNA3/HPV16E2 and anti-HPV16E2 antibody in the current study would provide a powerful tool for function research of HPV gene in cell level;
Finally,pCDNA3/HPV16E2 effects on SiHa cells growth number in detection.
pcDNA3/HPV16E2 was transfected into SiHa cells and E2 protein was expressed.The HPV16E6 mRNA expression and SiHa cell growth were detected.RT-PCR and Western blotting result respectively showed that in SiHa cells E2 mRNA and protein were expressed.In HPV16E2 gene transfection test, the pCDNA3/HPV16E2 group had significant lower HPV16E6 mRNA level and cell growth number than control group(P<0.05).
引文
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