本文選題：SARS + S1��； 參考：《河北師范大學(xué)》2006年碩士論文

【摘要】：SARS冠狀病毒主要蛋白質(zhì)包括RNA聚合酶蛋白、S蛋白、E蛋白、N蛋白等。其中S蛋白是負(fù)責(zé)病毒侵染的主要部分,S蛋白的差異可導(dǎo)致病毒宿主的更迭,并可使宿主產(chǎn)生感冒、腹膜炎、腸胃炎等多種疾病。S蛋白同時也是影響冠狀病毒侵染宿主程度的主要成分。本文主要研究了SARS冠狀病毒S1基因的原核表達(dá),并對表達(dá)蛋白進(jìn)行初步純化,利用純化的重組蛋白建立了檢測SARS抗體的ELISA方法。另外還構(gòu)建了S1基因的真核表達(dá)載體。為今后核酸疫苗的研制奠定了基礎(chǔ)。 首先以含有S1基因的質(zhì)粒為模板,對S1基因進(jìn)行PCR擴(kuò)增,將擴(kuò)增的基因片段克隆到pGEM T-Easy載體,經(jīng)雙酶切后進(jìn)行序列測定和同源性分析。在此基礎(chǔ)上構(gòu)建了S1基因的重組表達(dá)質(zhì)粒pGEX-4T1-S1,轉(zhuǎn)化宿主菌BL21(DE3)。表達(dá)的S1蛋白經(jīng)SDS-PAGE分析,在56KD處有一特異表達(dá)條帶。激光密度掃描結(jié)果表明,融合蛋白占細(xì)菌總蛋白的34.36%。經(jīng)Western blot檢測,表達(dá)的蛋白能夠被SARS陽性血清所識別,為SARS病毒感染診斷和SARS抗體檢測方法的建立奠定了基礎(chǔ)。 將表達(dá)的S1蛋白初步純化后作為抗原,建立了檢測SARS病毒抗體的間接ELISA方法。對ELISA的反應(yīng)條件進(jìn)行了研究,結(jié)果表明蛋白抗原的最適包被濃度為5.0μg/ml,最適包被條件選37℃孵育一小時然后4℃過夜,HRP-山羊抗人IgG的工作濃度為1:400。其特異性較好,患者血清未與正常血清發(fā)生交叉反應(yīng)。建立的間接ELISA為我國進(jìn)行SARS病毒的診斷和血清學(xué)檢測提供了一種技術(shù)手段,并為SARS檢測試劑盒的研制與開發(fā)奠定了前期工作基礎(chǔ)。 最后以含有S1基因質(zhì)粒為模板,對S1基因進(jìn)行PCR擴(kuò)增,序列分析正確后構(gòu)建了真核表達(dá)質(zhì)粒pVAX-S1,轉(zhuǎn)染Hela細(xì)胞后的免疫熒光檢測結(jié)果表明重組質(zhì)粒在體外細(xì)胞中得到表達(dá),為SARS DNA疫苗的研究奠定了一定的基礎(chǔ)。
[Abstract]:The main proteins of SARS coronavirus include RNA polymerase protein, S protein, protein E protein, protein N, etc. Among them, S protein is the main part responsible for virus infection. The difference of S protein can lead to the change of virus host, and can make the host produce colds and peritonitis. Many diseases, such as gastroenteritis, are also the main components of coronavirus infection. In this paper, the prokaryotic expression of S1 gene of SARS coronavirus was studied, and the expressed protein was preliminarily purified. A ELISA method for detection of SARS antibody was established by using the purified recombinant protein. In addition, the eukaryotic expression vector of S1 gene was constructed. It lays a foundation for the development of nucleic acid vaccine in the future. Firstly, the S1 gene was amplified by PCR using the plasmid containing S1 gene as the template. The amplified gene fragment was cloned into the pGEM T-Easy vector and sequenced and homologous analyzed by double enzyme digestion. On this basis, the recombinant expression plasmid pGEX-4T1-S1 of S1 gene was constructed, and the host strain BL21DE3 was transformed. The expressed S1 protein was analyzed by SDS-PAGE and there was a specific expression band in 56KD. The results of laser density scanning showed that the fusion protein accounted for 34.36% of the total bacterial protein. The expressed protein could be recognized by SARS positive serum by Western blot detection, which laid a foundation for the diagnosis of SARS virus infection and the establishment of SARS antibody detection method. The expressed S1 protein was initially purified as antigen, and an indirect ELISA method for detection of SARS virus antibody was established. The reaction conditions of ELISA were studied. The results showed that the optimal coating concentration of protein antigen was 5.0 渭 g / ml, and the optimal coating conditions were 37 鈩，

本文編號：1917584