【摘要】：尋找優(yōu)勢診斷抗原,一直是血吸蟲病診斷研究中的熱點(diǎn)。血吸蟲循環(huán)抗原因其具有反映活動性感染情況、評估蟲負(fù)荷和療效考核價(jià)值等特點(diǎn),一直倍受人們關(guān)注,尋找高敏感性和高特異性方法檢測循環(huán)抗原,也是血吸蟲病診斷研究長期面臨的挑戰(zhàn)。 鑒于本研究組在基因工程抗體方面的前期研究工作,本課題旨在通過已經(jīng)獲得的SIEA26-28kDa SjscFv,利用其對日本血吸蟲成蟲、蟲卵各發(fā)育階段的高特異性靶向免疫識別能力,充分發(fā)揮其作為探針的作用,從日本血吸蟲感染兔血清中,篩選血吸蟲循環(huán)抗原組分,并利用NanoLC-MS/MS質(zhì)譜以及生物信息學(xué)技術(shù)進(jìn)行初步分析,鑒定循環(huán)抗原的相關(guān)組分,以期尋找具有高效診斷價(jià)值的血吸蟲循環(huán)抗原,為建立高敏感性和高特異性檢測循環(huán)抗原方法奠定基礎(chǔ)。 研究目的 1.構(gòu)建原核質(zhì)粒pET43.1a/SjscFv,表達(dá)、純化與鑒定SjscFv目的蛋白。 2.利用SjscFv篩選日本血吸蟲感染兔血清中的循環(huán)抗原。 3.通過質(zhì)譜和生物信息學(xué)技術(shù)手段,分析循環(huán)抗原相關(guān)組分的結(jié)構(gòu)與功能,鑒定出具有診斷價(jià)值的候選抗原分子。 研究方法 1.原核質(zhì)粒的構(gòu)建及SjscFv蛋白的表達(dá)、純化與鑒定 根據(jù)SjscFv基因開放閱讀框架(ORF)設(shè)計(jì)上下游引物,以pET28a/SIEA26-28kDa-SjscFv質(zhì)粒為模板,構(gòu)建pET43.1a/SjscFv原核質(zhì)粒,經(jīng)PCR、雙酶切和DNA測序鑒定重組質(zhì)粒構(gòu)建是否成功。以最佳誘導(dǎo)條件大量培養(yǎng)BL21(DE3)大腸桿菌表達(dá)SjscFv目的蛋白,Western blot鑒定該蛋白活性,Ni-柱層析法純化蛋白并測定蛋白濃度。 2.日本血吸蟲感染兔血清中循環(huán)抗原的篩選 通過SDS-PAGE和Native-PAGE凝膠電泳、Western Blot技術(shù),利用pET43.1a/SjscFv蛋白識別日本血吸蟲感染兔血清中的循環(huán)抗原相關(guān)組分,尋找感染陽性兔血清與重組蛋白特異結(jié)合的抗原條帶。通過電泳收集特異條帶并進(jìn)行電洗脫純化。同時,將純化抗原與佐劑等體積混勻后免疫新西蘭兔,制備循環(huán)抗原相關(guān)組分免疫血清,采用血吸蟲成蟲和感染兔肝制作石蠟切片,進(jìn)行免疫熒光組化定位。 3.NanoLC-ESI-MS/MS質(zhì)譜技術(shù)分析SjscFv識別的血吸蟲感染兔血清中循環(huán)抗原的組分；通過生物信息學(xué)技術(shù)分析循環(huán)抗原相關(guān)組分的結(jié)構(gòu)與功能。 研究結(jié)果 1.成功構(gòu)建了原核質(zhì)粒pET43.1a/SjscFv,經(jīng)PCR、雙酶切及DNA測序結(jié)果均證實(shí)所構(gòu)建質(zhì)粒pET43.1a/SjscFv中基因片段與目的片段一致。誘導(dǎo)表達(dá)pET43.1a/SjscFv工程菌,在約91kDa處獲得大量表達(dá)蛋白,優(yōu)化的最佳表達(dá)條件為：IPTG濃度為0.05mmol/L,溫度為28℃,時間為過夜。采用Western Blot分析證實(shí)表達(dá)蛋白與目的蛋白一致。經(jīng)鎳柱層析法純化后,SIEA26-28kDa-SjscFv蛋白濃度可達(dá)到0.339mg/mL。 2.采用SIEA26-28kDa-SjscFv進(jìn)行Western Blot分析,發(fā)現(xiàn)SIEA26-28kDa-SjscFv可識別日本血吸蟲感染2周-10周的陽性兔血清中的95KDa循環(huán)抗原相關(guān)組分。通過Native-PAGE證實(shí)該分子量約為95kDa。Western Blot和免疫熒光定位顯示該循環(huán)抗原相關(guān)組分來源于成蟲體表,而在蟲卵中未出現(xiàn),提示其與血吸蟲感染早期的階段性抗原相關(guān)。 3.通過SDS-PAGE電泳獲得的循環(huán)抗原反應(yīng)條帶,經(jīng)切膠電洗脫分離純化后,采用NanoLC-ESI-MS/MS質(zhì)譜技術(shù)進(jìn)行分析,共鑒定出六種與血吸蟲循環(huán)抗原相關(guān)的組分分別為：heat shock protein, actin, Importinβ1,Na+/K+transporting ATPase subunit alpha,SjCHGC04905protein,SjCHGC06108protein。Importin-β1作生物信息學(xué)分析,推測它主要具有HEAT repeat結(jié)構(gòu)域、IBB-N結(jié)構(gòu)域和ARM repeat結(jié)構(gòu)域,α螺旋折疊成的三級結(jié)構(gòu)可伸展或收縮,無跨膜區(qū),存在豐富的抗原表位,預(yù)測其為較好的診斷候選分子。 結(jié)論 1.成功構(gòu)建原核重組質(zhì)粒pET43.1a/SjscFv并大量表達(dá)和純化SjscFv重組蛋白； 2.篩選與鑒定出六種與血吸蟲循環(huán)抗原相關(guān)的組分,該抗原在日本血吸蟲成蟲體壁大量表達(dá),推測其與血吸蟲感染早期的階段性抗原相關(guān)； 3.Importin-β1蛋白的結(jié)構(gòu)與功能分析推測它是一良好的診斷候選抗原分子。
[Abstract]:The search for superiority diagnostic antigen has been a hot spot in the diagnosis and research of schistosomiasis . It has been paid more attention to the diagnosis of schistosomiasis because it has the characteristics of reflecting active infection , evaluating the value of insect load and curative effect , and finding high sensitivity and high specificity method to detect circulating antigen . It is also a long - term challenge for the diagnosis of schistosomiasis .

In view of the previous research work on genetic engineering antibody , the aim of this study was to use the SIEA26 - 28kDa SjscFv which has been obtained to target the high - specific target immune recognition ability of Schistosoma japonicum adult and egg development stage , and to use the NanoLC - MS / MS mass spectrometry and bioinformatics technology to analyze the circulating antigen of Schistosoma japonicum , and to identify the relevant components of circulating antigen , so as to find the circulating antigen of Schistosoma japonicum with high efficiency diagnostic value , and lay the foundation for the establishment of high sensitivity and high specificity detection cycle antigen method .

Purpose of study

1 . Construction of prokaryotic plasmid pET43.1a / SjscFv , expression , purification and identification of SjscFv target protein .

2 . Using SjscFv to screen the circulating antigen of Schistosoma japonicum infected rabbit serum .

3 . By means of mass spectrometry and bioinformatics , the structure and function of circulating antigen - related components were analyzed , and the candidate antigen molecules with diagnostic value were identified .

Research Methods

1 . Construction of prokaryotic plasmid and expression , purification and identification of SjscFv protein

The prokaryotic plasmid pET43.1a / SjscFv was constructed by using pET28a / SIEA26 - 28kDa - SjscFv plasmid as template and pET28a / SIEA26 - 28kDa - SjscFv plasmid as template .

2 . Screening of circulating antigens in sera from rabbits infected with Schistosoma japonicum

By SDS - PAGE and Native - PAGE gel electrophoresis , Western Blot was used to identify the circulating antigen - related components of Schistosoma japonicum infected rabbit serum by using pET43 . 1a / SjscFv protein . The specific band was collected by electrophoresis and electroelution was carried out . At the same time , purified antigen was mixed with adjuvant and purified by electroelution .

3 . NanoLC - ESI - MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS / MS /
The structure and function of circulating antigen - related components were analyzed by bioinformatics .

Results of the study

1 . The prokaryotic plasmid pET43.1a / SjscFv was successfully constructed . The recombinant plasmid pET43.1a / SjscFv was constructed by PCR , double digestion and DNA sequencing . The recombinant plasmid pET43.1a / SjscFv was constructed . The expression of pET43 . 1a / SjscFv engineering bacteria was induced . The optimal expression conditions were as follows : IPTG concentration was 0.05 mmol / L , temperature was 28 鈩，

本文編號：2119109