本文選題：泡狀棘球蚴病 + 基因克隆��； 參考：《新疆醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：(1)通過基因工程技術(shù)克隆多房棘球絳蟲Em18抗原。(2)將HRP標(biāo)記在基因工程制備的Em18抗原上,利用雙抗原夾心法,制備泡狀棘球蚴快速診斷試劑盒。方法：(1)從多房棘球蚴中使用RNA提取試劑盒提取RNA,反轉(zhuǎn)錄成cDNA,通過克隆、構(gòu)建、測序,利用DNAman軟件對Em18基因特點(diǎn)進(jìn)行分析并構(gòu)建Em18核酸序列。用DNAman軟件設(shè)計(jì)引物,分別在5’端3’端添加EcoRI和Xhol酶切位點(diǎn),以pET28a/Em18原核表達(dá)質(zhì)粒為模板,PCR擴(kuò)增Em18基因片斷,經(jīng)酶切,轉(zhuǎn)化入大腸埃希菌表達(dá)載體BL21,酶切、PCR及測序鑒定其插入序列的正確性。擴(kuò)增出Em18抗原,測序鑒定序列正確。(2)經(jīng)IPTG誘導(dǎo)表達(dá)rEm18,洗脫純化,蔗糖濃縮,通過SDS-PAGE電泳和Western blot試驗(yàn)鑒定重組蛋白的表達(dá)水平。(3)采用HRP標(biāo)記基因重組抗原。(4)棋盤法篩選抗原包被濃度,酶標(biāo)抗原稀釋倍數(shù),待檢抗體稀釋倍數(shù),利用雙抗原夾心法制備Em18泡狀棘球蚴快速診斷試劑盒。結(jié)果：(1)成功克隆并構(gòu)建了pET28a/Em18原核表達(dá)質(zhì)粒,經(jīng)IPTG誘導(dǎo),SDS-PAGE電泳檢測表明pET28a/Em18重組蛋白得到成功表達(dá),在相對分子量為50kD處有表達(dá)條帶。(2) Western Blot分析顯示rEm18蛋白能被多房棘球蚴病人陽性血清識別,具有良好的抗原性。(3)通過酶結(jié)合物的評價指標(biāo)判斷HRP成功標(biāo)記抗原。將重組蛋白包被在酶聯(lián)板上,按照已篩選好的條件進(jìn)行組裝試劑盒,選擇待測樣本,檢測出試劑盒具有較高的敏感性和特異性。(4)通過單盲法利用配對χ2檢驗(yàn)比較雙抗原夾心法和斑點(diǎn)免疫膠體金法同金標(biāo)準(zhǔn)(手術(shù))的敏感性和特異性。P0.05,差異有統(tǒng)計(jì)學(xué)意義。結(jié)論：運(yùn)用基因克隆技術(shù)對Em18抗原進(jìn)行克隆,并成功表達(dá)、鑒定、濃縮和純化,成功使用HRP標(biāo)記重組抗原,利用棋盤滴定法篩選出試劑盒各因素最適條件,通過ELISA雙抗原夾心法原理組裝試劑盒,采用配對χ2檢驗(yàn)比較雙抗原夾心法和斑點(diǎn)免疫膠體金法同金標(biāo)準(zhǔn)的敏感性和特異性。χ2=5.1,P0.05,差異有統(tǒng)計(jì)學(xué)意義。
[Abstract]:Objective: to clone Em18 antigen of Echinococcus multilocularis by genetic engineering. To label HRP on Em18 antigen prepared by genetic engineering, and to prepare a rapid diagnostic kit for hydatid alveolar echinococcus by double antigen sandwich method. Methods RNA extraction kit was used to extract RNAs from echinococcus multilocularis and reverse transcribed into cDNA.Through cloning, construction and sequencing, the characteristics of Em18 gene were analyzed by DNAman software and the nucleic acid sequence of Em18 was constructed. DNAman software was used to design primers, EcoRI and Xhol sites were added at the 3 'end of 5' end respectively. The pET28a/Em18 prokaryotic expression plasmid was used as template to amplify the Em18 gene fragment, and was digested by enzyme. The recombinant plasmid BL21 was transformed into Escherichia coli expression vector BL21, and its insertion sequence was confirmed by restriction endonuclease polymerase chain reaction (PCR) and sequencing. Em18 antigen was amplified and sequenced to identify the correct sequence. It was induced by IPTG to express rEm18, eluted and purified, and sucrose was concentrated. The expression level of recombinant protein was identified by SDS-PAGE electrophoresis and Western blot test. The antigen coating concentration, dilution multiple of enzyme labeled antigen, dilution multiple of antibody to be detected were screened by using HRP labeled gene recombinant antigen. 4) chessboard method was used to screen the concentration of antigen, the dilution multiple of enzyme labeled antigen, and the dilution multiple of antibody to be detected. The rapid diagnostic kit of Em18 alveolar echinococcus was prepared by double antigen sandwich method. Results the prokaryotic expression plasmid of pET28a/Em18 was cloned and constructed successfully. IPTG induced SDS-PAGE electrophoresis showed that the recombinant pET28a/Em18 protein was successfully expressed. Western Blot analysis showed that the rEm18 protein could be recognized by the positive sera of patients with multilocularis echinococcosis, and had good antigenicity. The recombinant protein was coated on the enzyme linked plate, and the kit was assembled according to the selected conditions, and the samples to be tested were selected. The sensitivity and specificity of double antigen sandwich assay and dot immune colloidal gold standard (P0.05) were compared by paired 蠂 2 test with single blind method. The difference was statistically significant. Conclusion: the Em18 antigen was cloned by gene cloning technique and successfully expressed, identified, concentrated and purified. The recombinant antigen labeled with HRP was successfully used. The optimum conditions of each factor of the kit were screened by chessboard titration. The kit was assembled by ELISA double antigen sandwich method, and the sensitivity and specificity of double antigen sandwich method and dot immunocolloid gold method were compared by paired 蠂 2 test. The difference was statistically significant.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R446.6;R532.32

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