eRF3b羧基端部分多肽片段的分子克

发布时间：2018-08-21 09:19

【摘要】：目的:乙型病毒性肝炎是由乙型肝炎病毒(HBV)引起的一种在世界范围内广为流行的一种疾病,是已知各型肝炎中危害最严重的一种。我国是世界肝炎大国,大约有7亿人感染过乙肝,即总感染率为57%;有2300万慢性肝炎患者,9300万乙肝病毒携带者;每年因肝炎死亡的人数约有23万人[1]。鉴于上述原因,我国在乙肝的预防和治疗上也花费了很多费用,有报导可见每年防治乙肝的经费约500亿人民币[2,3]。尽管对于乙肝的研究很多,但是乙肝迄今为止也没有很好的治愈方法,所以乙肝对全国人民的健康水平和国家财政收入都造成了很大的威胁。因此,临床上迫切需要研究新的灵敏度和特异度都较高的诊断方法,并且发现新的有价值的生物标志物,争取做到早预防、早诊断、早治疗,改善病人情况。研究乙型病毒性肝炎机制及其制备eRF3b羧基端部分多肽片段多克隆抗体,具有重大意义。本室前期利用MALDI-TOF MS技术发现4210Da蛋白为“GSPT2,真核肽链释放因子GTP结合亚单位(eRF)”,也就是eRF3b裂解后的一部分—37肽。GSPT2编码eRF3b,具有参与蛋白合成中止的功能。eRF3b在肝脏感染乙肝病毒后的免疫反应过程中发挥了极其重要的作用,4210Da多肽作为其中一部分肽段而不断出现在血液中,而且表达量在不同肝病组中差异明显,提示该蛋白可以作为急性乙肝(AHB)转为慢性乙肝(CHB)以及从慢性乙肝(CHB)患者中筛检肝癌(HCC)患者的生物学标志物[1]。由于本研究是针对GSPT2氨基端第1421—1531位碱基进行的研究,其间含有111个碱基,因此称其基因为GSPT2-111,其对应的蛋白称为eRF3b-37。本研究的目的是制备融合蛋白GST-eRF3b-37兔多克隆抗体和GST-eRF3b-37大鼠多克隆抗体,为今后研究乙肝标志物提供了依据,同时为乙肝分子流行病学研究奠定了基础。方法: (1)GSPT2-111的分子克隆。采用胍盐酸法[14]从外周血中提取人基因组DNA,运用聚合酶链式反应(PCR)扩增获得大量GSPT2-111基因片段。将GSPT2-111基因片段通过BamH I和EcoR I两个酶切位点连接至pGEX-4T-1(GST)这一表达载体上,并应用碱裂解法[14]提取质粒,用BamH I和EcoR I双酶切后进行质粒PCR鉴定及测序分析。 (2)融合蛋白GST-eRF3b-37的表达及可溶性鉴定。将GSPT2-111基因片段克隆到pGEX-4T-1(GST)这一表达载体上,转入大肠杆菌DH5α后,经1.0 mM IPTG诱导后获得带有GST标签的GST-eRF3b-37融合蛋白,通过10% SDS-PAGE电泳,检测融合蛋白表达及可溶性。 (3)融合蛋白GST-eRF3b-37的纯化。大量表达GST-eRF3b-37融合蛋白,通过GST SefinoseTM Resin纯化带有GST标签的GST-eRF3b-37融合蛋白,然后进行10% SDS-PAGE电泳,检测GST-eRF3b-37融合蛋白纯化情况。 (4)多克隆抗体的制备。用融合蛋白GST-eRF3b-37作为抗原对新西兰大白兔和大鼠进行免疫,取1ml PBS(其中含有1mg抗原)和等体积的完全弗氏佐剂乳化后在动物背部6个不同的部位进行皮下注射,每只家兔每个部位注射约300μl抗原溶液,每只大鼠每个部位注射约30μl抗原溶液。每两周免疫一次,连续4次。首次免疫用完全弗氏佐剂,后三次免疫用不完全弗氏佐剂。 (5)多克隆抗体的检测及纯化。通过蛋白斑点杂交试验检测免疫动物血清中是否存在融合蛋白GST-eRF3b-37抗体。采用Protein A亲和层析柱对融合蛋白抗体进行纯化,纯化后的抗体运用间接ELISA法测定抗体效价,并通过Western-blot方法检测融合蛋白GST-eRF3b-37抗体的特异性。结果: (1)GSPT2-111的分子克隆。本研究从人外周血中成功克隆出GSPT2-111基因片段111bp。GSPT2-111编码37个氨基酸,推测其相对分子质量为4.1kDa。 (2)融合蛋白GST-eRF3b-37的表达、可溶性鉴定及纯化。含重组质粒pGEX-4T-1-GSPT2-111大肠杆菌DH5α经1.0 mM IPTG诱导,出现一分子量约30kDa的新蛋白表达,与GST(分子量为26kDa)和eRF3b-37(分子量约为4.1kDa)的理论分子量相符,并且为可溶性蛋白。通过GST SefinoseTM Resin纯化出足够免疫2只新西兰大白兔和10只大鼠的融合蛋白,用融合蛋白对动物进行为期两个月的免疫。 (3)多克隆抗体的检测及纯化。四次免疫后运用蛋白斑点杂交试验检测出血清中存在融合蛋白GST-eRF3b-37抗体,再通过间接ELISA法测试抗体效价,纯化前的兔抗体效价大于51,200,大鼠抗体效价大于12,800。收集动物血清进行Protein A亲和层析柱纯化,纯化后的兔抗体效价大于384,000,IgG为60mg/ml。纯化后的大鼠抗体效价大于25,600,IgG为25mg/ml。Western-blot结果显示,无论是兔抗体按1:6000稀释,还是鼠抗体按1:3000稀释,纯化出的抗体均可检测出清晰的分子量约为30kDa的GST-eRF3b-37融合蛋白的条带。结论: (1)克隆出了GSPT2-111基因片段,并且构建了重组质粒pGEX-4T-1-GSPT2-111。 (2)表达并且纯化出GST-eRF3b-37融合蛋白。 (3)制备并且纯化出兔抗人GST-eRF3b-37多克隆抗体和大鼠抗人GST-eRF3b-37多克隆抗体。
[Abstract]:Objective: Hepatitis B virus (HBV) is a worldwide epidemic disease caused by hepatitis B virus (HBV), which is one of the most harmful types of hepatitis known in the world. Carriers: About 230,000 people die of hepatitis every year. For these reasons, our country also spends a lot of money on prevention and treatment of hepatitis B. It has been reported that the annual cost of prevention and treatment of hepatitis B is about 50 billion yuan [2,3]. Although there are many studies on hepatitis B, there is no good cure for hepatitis B, so far. Hepatitis B poses a great threat to the health level of the people and the national financial revenue. Therefore, it is urgent to study new diagnostic methods with high sensitivity and specificity, and find new valuable biomarkers, so as to achieve early prevention, early diagnosis, early treatment and improve the patient's condition.
It is of great significance to study the mechanism of viral hepatitis B and to prepare polyclonal antibodies against the carboxyl-terminated polypeptide fragments of eRF3b. In the previous study, MALDI-TOF MS technique was used to identify 4210Da protein as "GSPT2, Eukaryotic Peptide Chain Releasing Factor GTP Binding Subunit (eRF)", which is a part of the cleavage of eRF3b-37 peptide.GSPT2 encodes eRF3b. ERF3b plays an important role in the immune response to hepatitis B virus infection. 4210Da polypeptide, as a part of the peptide, is constantly present in the blood. The expression of eRF3b is significantly different in different groups of liver diseases, suggesting that it can be used as an acute hepatitis B (AHB) to change to chronic hepatitis B. (CHB) and biomarkers [1]. in patients with liver cancer (HCC) screened from chronic hepatitis B (CHB).
The aim of this study is to prepare the rabbit polyclonal antibody against GST-eRF3b-37 and the rat polyclonal antibody against GST-eRF3b-37, which will be used as a marker for the future study of hepatitis B. It provided a basis for the study, and laid the foundation for the molecular epidemiological study of hepatitis B.
Method:
(1) Molecular cloning of GSPT2-111. Human genomic DNA was extracted from peripheral blood by guanidine hydrochloric acid method [14] and a large number of GSPT2-111 gene fragments were amplified by polymerase chain reaction (PCR). The GSPT2-111 gene fragments were linked to the expression vector pGEX-4T-1 (GST) by BamH I and EcoR I, and extracted by alkaline lysis [14]. Plasmids were digested with BamH I and EcoR I, then plasmid PCR was identified and sequenced.
(2) Expression and solubility identification of GST-eRF3b-37 fusion protein. GSPT2-111 gene fragment was cloned into pGEX-4T-1 (GST) expression vector. After transfection into E. coli DH5a, GST-eRF3b-37 fusion protein with GST tag was obtained by 1.0 mM IPTG induction. The expression and solubility of GST-eRF3b-37 fusion protein were detected by 10% SDS-PAGE electrophoresis.
(3) Purification of fusion protein GST-eRF3b-37. GST-eRF3b-37 fusion protein was expressed in large quantities. GST-eRF3b-37 fusion protein with GST tag was purified by GST SefinoseTM Resin. The purified GST-eRF3b-37 fusion protein was detected by 10% SDS-PAGE electrophoresis.
(4) Preparation of polyclonal antibodies. New Zealand white rabbits and rats were immunized with fusion protein GST-eRF3b-37 as antigen. 1 ml PBS (containing 1 mg antigen) and an equivalent volume of complete Freund's adjuvant were emulsified and subcutaneously injected into 6 different parts of the back of the animal. Each rabbit was injected with about 300 ml antigen solution at each part, each large. About 30 ml antigen solution was injected into each part of the mice every two weeks for four consecutive times. Complete Freund's adjuvant was used for the first time and incomplete Freund's adjuvant was used for the third time.
(5) Detection and purification of polyclonal antibodies. Detection of fusion protein GST-eRF3b-37 antibody in serum of immunized animals by dot blot hybridization. Purification of fusion protein antibody by Protein A affinity chromatography column. Indirect ELISA was used to determine antibody titer and Western blot was used to detect fusion protein antibody. Specificity of protein GST-eRF3b-37 antibody.
Result:
(1) Molecular cloning of GSPT2-111. In this study, 111bp.
(2) Expression, solubility identification and purification of fusion protein GST-eRF3b-37. Induced by 1.0 mM IPTG, the recombinant plasmid pGEX-4T-1-GSPT2-111 of E. coli DH5a was expressed with a molecular weight of about 30 kDa, which was consistent with the theoretical molecular weight of GST (26 kDa) and eRF3b-37 (4.1 kDa) and was a soluble protein.
Fusion proteins were purified from GST SefinoseTM Resin to immunize 2 New Zealand white rabbits and 10 rats for two months.
(3) Detection and purification of polyclonal antibodies. After four immunizations, the fusion protein GST-eRF3b-37 was detected by dot blot hybridization. The antibody titer was tested by indirect ELISA. The rabbit antibody titer was more than 51,200 and the rat antibody titer was more than 12,800 before purification. The titer of purified rabbit antibody was higher than 384,000 and the IgG was 60mg/ml. The titer of purified rat antibody was higher than 25,600 and the IgG was 25mg/ml. Western-blot results showed that the purified antibody could detect the GST-eRF3b-37 fusion protein with a clear molecular weight of about 30kDa, whether the rabbit antibody was diluted at 1:6000 or the mouse antibody was diluted at 1:3000. The strip.
Conclusion:
(1) cloned the GSPT2-111 gene fragment and constructed the recombinant plasmid pGEX-4T-1-GSPT2-111..
(2) expression and purification of GST-eRF3b-37 fusion protein.
(3) Rabbit anti-human GST-eRF3b-37 polyclonal antibody and rat anti-human GST-eRF3b-37 polyclonal antibody were prepared and purified.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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