本文選題：剛地弓形蟲 + 重組致密顆粒蛋白GRA5��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的原核表達(dá)和鑒定剛地弓形蟲致密顆粒蛋白5(GRA5),以期獲得大量與天然抗原活性相似的弓形蟲重組GRA5蛋白抗原,將獲得的重組蛋白進(jìn)行純化,應(yīng)用純化的GRA5重組蛋白抗原包被在96孔板上,建立ELISA法檢測(cè)弓形蟲感染。方法從GenBank中查到弓形蟲GRA5基因序列,根據(jù)基因序列設(shè)計(jì)合成一對(duì)引物,用RT-PCR方法將GRA5基因擴(kuò)增,構(gòu)建pET28a-GRA5原核表達(dá)載體,雙核酸內(nèi)酶切及序列測(cè)定進(jìn)行鑒定,IPTG誘導(dǎo)pET28a-GRA5轉(zhuǎn)化的BL21/DE3菌,SDS-PAGE和Western-blotting分析表達(dá)產(chǎn)物并鑒定弓形蟲GRA5是否表達(dá)。建立以純化的重組蛋白的間接ELISA法,檢測(cè)收集樣本血清中弓形蟲特異性抗體。結(jié)果成功構(gòu)建剛地弓形蟲GRA5基因原核表達(dá)質(zhì)粒,PCR反應(yīng)擴(kuò)增出為363 bp大小的GRA5基因,所構(gòu)建的pET28a-GRA5原核表達(dá)載體經(jīng)雙酶切顯示插入片段大小與上相符,DNA測(cè)序結(jié)果表明與GenBank中錄入的GRA5基因經(jīng)Blast比對(duì)序列同源性100%,原核細(xì)胞表達(dá)的該重組蛋白在SDS-PAGE和Western blot中均有顯示(約14 ku)。將重組GRA5蛋白作為抗原包被在96孔板中,建立了檢測(cè)弓形蟲感染的ELISA方法,ELISA法經(jīng)過優(yōu)化后最終確定:最佳抗原包被濃度為10ug/ml、最佳條件為在37℃作用2h后,在4℃包被過夜、弓形蟲血清最佳稀釋度為1:25,最佳封閉條件為用5%脫脂奶粉在37℃作用2h,二抗最佳工作濃度為1:20000,底物的最佳反應(yīng)時(shí)間為20min。本實(shí)驗(yàn)建立的ELISA法檢測(cè)的100例弓形蟲感染病人(血清學(xué)陽性)血清中有73例呈陽性,陽性率為73%(73/100),其中40例IgG陽性標(biāo)本的陽性率為72.5%(29/40),30例IgM陽性標(biāo)本的陽性率為53.3%(16/30),30例IgG、IgM均陽性標(biāo)本的陽性率為93.3%(28/30),30例陰性血清標(biāo)本的陽性率僅為6.7%(2/30)
[Abstract]:Objective to express and identify Toxoplasma gondii dense granuloprotein 5 (GRA5) in order to obtain a large number of recombinant GRA5 protein antigens similar to those of natural antigens, and to purify the recombinant proteins obtained from Toxoplasma gondii (Toxoplasma gondii).The purified GRA5 recombinant protein antigen was coated on 96 well plate to establish ELISA assay for detection of Toxoplasma gondii infection.Methods the GRA5 gene sequence of Toxoplasma gondii was identified from GenBank. A pair of primers were designed and synthesized according to the sequence of Toxoplasma gondii. The GRA5 gene was amplified by RT-PCR method to construct the prokaryotic expression vector of pET28a-GRA5.DNA endonuclease digestion and sequencing were performed to identify the expression products of BL21/DE3 transformed by BL21/DE3 induced by IPTG and Western-blotting analysis, and to identify the expression of Toxoplasma gondii GRA5 (Toxoplasma gondii).An indirect ELISA assay was established to detect Toxoplasma gondii specific antibodies in serum of purified recombinant proteins.Results We successfully constructed the prokaryotic expression plasmid of Toxoplasma gondii GRA5 gene and amplified the 363bp GRA5 gene.The construction of pET28a-GRA5 prokaryotic expression vector was detected by double enzyme digestion. The result of sequencing showed that the inserted GRA5 gene was homologous to the GRA5 gene input in GenBank by Blast alignment sequence. The recombinant protein expressed by prokaryotic cells was expressed in SDS-PAGE and Western blot.All of them were displayed (about 14 kus).The recombinant GRA5 protein was coated in 96-well plate. A ELISA method for detecting Toxoplasma gondii infection was established. The optimized Elisa method was determined as follows: the best antigen coating concentration was 10ugrml, and the best condition was that the antigen was coated overnight at 4 鈩，

本文編號(hào)：1759847