本文關(guān)鍵詞： 弓形蟲 SAG4 克隆表達 免疫反應(yīng)性　出處：《廣西醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:獲取弓形蟲表面抗原SAG4目的基因序列,預(yù)測其編碼蛋白作為候選疫苗的可行性。構(gòu)建重組表達質(zhì)粒pET28a(+)-SAG4,在大腸埃希菌表達系統(tǒng)中表達SAG4,并檢測表達產(chǎn)物的免疫反應(yīng)性,為弓形蟲病的免疫診斷和疫苗研制奠定基礎(chǔ)。 方法:提取RH株弓形蟲的基因組DNA,根據(jù)GenBank中RH標準株弓形蟲的SAG4基因序列設(shè)計引物,PCR法體外擴增獲取目的基因片段。PCR產(chǎn)物經(jīng)測序鑒定及膠回收純化后,連接入pMD19-T載體中以構(gòu)建重組克隆質(zhì)粒pMD19-T-SAG4。用限制性內(nèi)切酶NcoⅠ、XhoⅠ從pMD19-T-SAG4中切下目的基因,亞克隆入pET28a(+)載體中構(gòu)建重組表達質(zhì)粒pET28a(+)-SAG4,并采用雙酶切反應(yīng)及測序方法鑒定重組體。用生物信息學(xué)方法對重組SAG4基因編碼序列進行同源性分析以及二級結(jié)構(gòu)和抗原表位的預(yù)測。將重組表達質(zhì)粒pET28a(+)-SAG4轉(zhuǎn)入大腸埃希菌BL21中,經(jīng)IPTG誘導(dǎo)表達后,用SDS-PAGE及蛋白質(zhì)印跡(Western blotting)技術(shù)分析鑒定表達產(chǎn)物的免疫反應(yīng)性。 結(jié)果:PCR體外擴增獲得的目的基因片段長537bp,經(jīng)BLAST同源性分析顯示該序列與RH株弓形蟲SAG4基因cDNA具有高度同源性,而與其他生物的同源性較低。雙酶切反應(yīng)及測序分析顯示,SAG4目的基因已被正確地連接入重組克隆質(zhì)粒pMD19-T-SAG4和重組表達質(zhì)粒pET28a(+)-SAG4中。BLAST比對及疏水性分析表明,重組基因編碼產(chǎn)物為RH株弓形蟲SAG4,是一種外膜蛋白;二級結(jié)構(gòu)預(yù)測該產(chǎn)物可形成多個無規(guī)卷曲和β-轉(zhuǎn)角區(qū)域;親水性、可及性、柔韌性、極性參數(shù)及抗原表位的綜合預(yù)測顯示,其氨基酸序列的第40,80,120,130位點附近的抗原性指數(shù)較高。重組表達質(zhì)粒pET28a(+)-SAG4經(jīng)IPTG誘導(dǎo)后,在BL21中以包涵體的形式穩(wěn)定表達SAG4。SDS-PAGE檢測可發(fā)現(xiàn)一特異性蛋白區(qū)帶,分子量約為18.74 ku,Western blotting分析顯示該條帶能被弓形蟲慢性感染血清識別。 結(jié)論:首次從RH株弓形蟲中克隆出緩殖子期特異性抗原SAG4基因編碼序列;成功構(gòu)建了SAG4目的基因的重組克隆質(zhì)粒pMD19-T-SAG4與重組表達質(zhì)粒pET28a(+)-SAG4;生物信息學(xué)分析技術(shù)預(yù)測出重組SAG4基因編碼產(chǎn)物具有良好的抗原性;重組表達質(zhì)粒pET28a(+)-SAG4在大腸埃希菌BL21中以包涵體形式高效表達了SAG4目的基因片段,該產(chǎn)物具有特異的免疫反應(yīng)性。以此推測出SAG4不但是具有良好應(yīng)用前景的弓形蟲候選疫苗分子,還有望應(yīng)用于弓形蟲慢性感染的檢測。
[Abstract]:Objective: to obtain the target gene sequence of Toxoplasma gondii surface antigen (SAG4) and predict the feasibility of its coding protein as a candidate vaccine, and construct the recombinant expression plasmid pET28a (pET-SAG4). SAG4 was expressed in Escherichia coli expression system, and the immunoreactivity of the expressed product was detected, which laid a foundation for immunological diagnosis and vaccine development of toxoplasmosis. Methods: the genomic DNA of Toxoplasma gondii was extracted from RH strain and primers were designed according to the SAG4 gene sequence of RH standard strain Toxoplasma gondii in GenBank. The target gene fragment was amplified by PCR in vitro. The product was sequenced and purified by gel recovery. The recombinant plasmid pMD19-T-SAG4 was constructed by ligation into pMD19-T vector. Nco 鈪，

本文編號：1449240