發(fā)布時間：2018-05-01 11:18

  本文選題：風疹病毒 + E1特異肽段��； 參考：《南京醫(yī)科大學》2009年碩士論文

【摘要】：風疹病毒(rubellavirus,RV),又稱德國麻疹病毒,是披膜病毒科風疹病毒屬的唯一成員,與披膜科的其它病毒無交叉抗原。RV是TORCH綜合征病原體(巨細胞病毒、單純皰疹病毒、風疹病毒、弓形蟲)之一,是重要的胎兒致畸因子,可以通過胎盤感染胎兒,導致新生兒患上先天性風疹綜合征(congenitalrubellasyndrome,CRS),可表現(xiàn)為白內(nèi)障、耳聾、心臟病或智力發(fā)育障礙。孕婦感染RV越早,新生兒患CRS的機率越大。RV也是男性不育的影響因子,男性生殖系統(tǒng)感染TORCH,可引起睪丸炎、附睪炎、前列腺炎等,以及男性不育。 目前,免疫學檢測手段一直是廣泛應用于RV感染的臨床診斷方法,如血凝抑制試驗(HIA),特異性抗體IgM、IgG及RV蛋白抗原的檢測等。RV感染的篩查主要基于ELISA法檢測特異性的血清IgM型抗RV抗體,檢測試劑盒幾乎完全依賴于進口,價格昂貴,很難適合于臨床實驗室常規(guī)開展。抗RV抗體檢測結(jié)果的準確性依賴于相應的抗原制劑,因此,抗原的特異性決定了檢測結(jié)果的準確性。盡管RV的主要三種結(jié)構(gòu)蛋白——包膜糖蛋白E1、E2和衣殼蛋白C都有著較高的免疫原性,在人體內(nèi)可誘發(fā)強烈的抗體應答。其中E1包括RV的大部分血凝抑制區(qū)和免疫抗原表位,且經(jīng)研究表明E1基因序列株間變異程度與全株序列變異程度相近,而E2蛋白的生物學作用尚不清楚,衣殼蛋白C主要與病毒復制有關(guān),后者因存在交叉抗體應答限制了其作為診斷的特異性抗原。因此,RV的3種結(jié)構(gòu)蛋白中,E1蛋白最適合作為基因工程抗原用做免疫性診斷試劑和疫苗的研制。 本研究從麻疹、腮腺炎及風疹三聯(lián)減毒活疫苗中提取了RV的基因組RNA,將其逆轉(zhuǎn)錄為cDNA,便于對其基因組保存及進一步擴增目的基因。而后通過基因工程方法表達E1基因保守性好、免疫原性高的氨基端196-305位氨基酸的肽段E1-N。用PCR擴增得到編碼E1-N的基因片段,插入原核表達載體pGEX-2T質(zhì)粒中,構(gòu)建成表達E1-N的重組質(zhì)粒pGEX-2T/E1-N,經(jīng)雙酶切和測序鑒定證實后,將該質(zhì)粒轉(zhuǎn)化E.coliBL21菌株,經(jīng)異丙基-β-D硫代半乳糖苷(IPTG)誘導,分別于16℃和37℃下誘導表達融合蛋白,得到結(jié)果:在16℃誘導下表達的谷胱甘肽-S-轉(zhuǎn)移酶(GST)與E1-N的融合蛋白主要存在于上清中;而37℃誘導下的融合蛋白主要存在于包涵體中。通過谷胱甘肽瓊脂糖珠親和層析獲得純化的GST/E1-N融合蛋白。 通過以上研究,我們擴增的包膜糖蛋白的E1特異基因序列,經(jīng)測序證實與GenBank上所報道的基因序列一致,用IPTG誘導重組表達質(zhì)粒pGEX-2T/E1-N表達重組融合肽段時,IPTG的最佳濃度為1mmol/L,最佳誘導時間為5h,最佳誘導溫度為16℃。低溫誘導可以避免包涵體的形成,重組融合蛋白以可溶性形式表達于上清中,從而避免了對重組融合蛋白的變性和復性過程,大大簡化了操作。因此,本研究表達的E1特異蛋白為進一步研制特異性RV檢測試劑盒和開發(fā)新型基因工程疫苗提供了實驗依據(jù)和方法基礎(chǔ)。
[Abstract]:Rubella virus, also known as rubella virus, is the only member of the rubella virus genus of the family Erythroviridae. RV is the pathogen of TORCH syndrome (cytomegalovirus, herpes simplex virus, rubella virus). Toxoplasma gondii), one of the important fetal teratogenic factors, can infect the fetus through the placenta and lead to congenital rubella syndrome (CCS), which can be characterized by cataract, deafness, heart disease or mental retardation. The earlier the pregnant woman infects RV, the greater the chance of neonatal CRS. RV is also the influence factor of male infertility. The male reproductive system infection with Torch can cause orchitis, epididymitis, prostatitis, and male infertility. At present, immunological detection has been widely used in the clinical diagnosis of RV infection. Such as hemagglutination inhibition test, detection of specific antibody IgG and RV protein antigen, and so on. The screening of RV infection is mainly based on ELISA method to detect the specific serum IgM type anti-RV antibody. The detection kit is almost completely dependent on imports, and the price is high. It is difficult to be used in clinical laboratory routine. The accuracy of anti-RV antibody detection depends on the corresponding antigens, so the specificity of antigens determines the accuracy of the detection results. Although the three main structural proteins of RV, the envelope glycoprotein E1E _ 2 and capsid protein C, have high immunogenicity, they can induce a strong antibody response in human body. Among them, E1 includes most of hemagglutination inhibition region and immune antigen epitope of RV. The results show that the variation degree of E1 gene sequence is similar to that of whole plant sequence, but the biological function of E2 protein is not clear. Capsid protein C is mainly associated with viral replication, which is restricted by cross-antibody responses as a specific antigen for diagnosis. Therefore, among the three structural proteins of RV, the E 1 protein is the most suitable for the development of immunological diagnostic reagent and vaccine as a genetic engineering antigen. In this study, the genomic RNAs of RV were extracted from live attenuated measles, mumps and rubella vaccine. Then we expressed the amino acid peptide E1-Nof the amino terminal of the amino terminal of 196-305 amino acid with high immunogenicity and good conservation of E1 gene by genetic engineering method. The gene fragment encoding E1-N was amplified by PCR and inserted into the prokaryotic expression vector pGEX-2T plasmid. The recombinant plasmid pGEX-2T / E1-N was constructed and confirmed by double enzyme digestion and sequencing. The plasmid was transformed into E.coliBL21 strain. The fusion protein was induced by isopropyl- 尾 -D-galactothioside (IPTG) at 16 鈩，

本文編號：1829119