本文關(guān)鍵詞：SARS相關(guān)冠狀病毒S1基因片段真核表達(dá)載體的構(gòu)建及其免疫活性測定　出處：《南華大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的:根據(jù)GeneBank登錄的BJ01株SARS-CoV核酸序列合成801bp S1基因片段(22382bp～23182bp),構(gòu)建SARS-CoV相關(guān)冠狀病毒S1基因片段真核表達(dá)載體,并肌注免疫BALB/c小鼠,觀察小鼠所產(chǎn)生的體液免疫和細(xì)胞免疫應(yīng)答水平,從而為SARS-CoV生物學(xué)活性的研究及核酸疫苗的研制提供實(shí)驗(yàn)依據(jù)。 方法:用PRIMER5.0引物設(shè)計(jì)軟件設(shè)計(jì)引物,聚合酶鏈反應(yīng)(PCR)擴(kuò)增S1基因片段801bp。將PCR產(chǎn)物純化后與pUCm-T載體連接,經(jīng)藍(lán)白斑篩選、雙酶切鑒定及序列分析后,亞克隆至pcDNA3.1(+)真核表達(dá)載體中;陽性克隆經(jīng)雙酶切鑒定后轉(zhuǎn)染HeLa細(xì)胞,免疫細(xì)胞化學(xué)法與Western-Blotting鑒定其在真核細(xì)胞中的表達(dá);以pcDNA3.1(+)/S1肌注免疫6周齡BALB/C小鼠,PCR與免疫組化法檢測SARS相關(guān)冠狀病毒S1基因在小鼠股四頭肌的表達(dá),ELISA法檢測抗SARS-CoV IgG,MTT法檢測免疫小鼠的T細(xì)胞增殖活性,并檢測免疫小鼠脾淋巴細(xì)胞的IFN-γ水平。 結(jié)果:擴(kuò)增的SARS-CoVS1基因片段801bp亞克隆至pcDNA3.1(+)/S1后,免疫細(xì)胞化學(xué)試驗(yàn)與Western-Blotting結(jié)果表明pcDNA3.1(+)/S1可以在HeLa細(xì)胞中表達(dá)大小約為32KD的S1蛋白。免疫組織化學(xué)結(jié)果顯示pcDNA3.1(+)/S1免疫組小鼠股四頭肌細(xì)胞內(nèi)表達(dá)了SARS-CoV S1蛋白。免疫后小鼠的體液與細(xì)胞免疫應(yīng)答水平均隨免疫時間增強(qiáng)。隨著免疫次數(shù)的增加和接種時間的延續(xù),pcDNA3.1(+)/S1免疫組小鼠體內(nèi)抗SARS-CoV IgG量明顯升高,抗體滴度達(dá)1∶2000。T淋巴細(xì)胞增殖實(shí)驗(yàn)表明,pcDNA3.1(+)/S1注射組細(xì)胞加入刺激物后,細(xì)胞成團(tuán)生長,pET-22b/S1重組蛋白刺激組較PHA刺激組細(xì)胞增殖明顯活躍,各組間有顯著性差異(采用方差分析,F=12.045,P=0.000,P0.05);細(xì)胞因子IFN-γ檢測
[Abstract]:Objective: to synthesize 801bp S1 gene fragment (22382bphp) according to the nucleic acid sequence of BJ01 strain SARS-CoV entered by GeneBank. The eukaryotic expression vector of S1 gene fragment of SARS-CoV associated coronavirus was constructed and BALB/c mice were immunized intramuscularly to observe the level of humoral immunity and cellular immune response. It provides experimental basis for the study of SARS-CoV biological activity and the development of nucleic acid vaccine. Methods: the primers were designed with PRIMER5.0 primer design software. The S1 gene fragment was amplified by polymerase chain reaction (PCR). The PCR product was purified and ligated with pUCm-T vector. Subcloned into eukaryotic expression vector pcDNA3.1 (); The positive clones were identified by double enzyme digestion and transfected into HeLa cells. Their expression in eukaryotic cells was identified by immunocytochemistry and Western-Blotting. The expression of SARS associated coronavirus S1 gene in the quadriceps femoris of BALB/C mice at 6 weeks of age was detected by pcDNA3.1 (intramuscular injection) and immunohistochemical method. The activity of T cell proliferation and the level of IFN- 緯 in spleen lymphocytes of immunized mice were detected by ELISA assay. Results: the amplified SARS-CoVS1 gene fragment 801bp was subcloned into pcDNA3.1 (P / S1). The results of immunocytochemistry and Western-Blotting showed that pcDNA3.1 (). RS1 could express about 32KD S1 protein in HeLa cells. Immunohistochemical results showed that pcDNA3.1 (). SARS-CoV was expressed in quadriceps femoris cells of mice immunized with rS1. S1 protein. The humoral and cellular immune response levels of mice after immunization increased with the immune time, with the increase of immunization times and the extension of inoculation time. The antibody titer was 1: 2000.T lymphocyte proliferation test showed that the amount of anti SARS-CoV IgG was significantly increased in mice immunized with pcDNA3.1 (P / S 1). After the cells were injected with pcDNA3.1, the proliferation of pET-22bS1 recombinant protein stimulated by pET-22bS1 recombinant protein was more active than that of PHA group. There was significant difference among the three groups (using the analysis of variance analysis of variance (AANOVA) 12.045A, P0. 000P0. 05); Cytokine IFN- 緯 detection
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R373

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本文編號：1371273