本文選題：粉塵螨 + .kDa溶菌酶(Bac)��； 參考：《中國(guó)人獸共患病學(xué)報(bào)》2016年09期

【摘要】：目的克隆表達(dá)粉塵螨13.8kDa溶菌酶(Bac)基因,純化鑒定蛋白的過(guò)敏原性,并進(jìn)行生物信息學(xué)分析。方法挑取經(jīng)純培養(yǎng)的粉塵螨,提取總RNA,根據(jù)已知基因序列設(shè)計(jì)引物,經(jīng)RT-PCR擴(kuò)增Bac基因片段,產(chǎn)物連入pMD-32T載體中。擴(kuò)增后,利用限制性內(nèi)切酶EcoRI和XhoI雙酶切將目的基因片段連接到Pet-44a表達(dá)載體上,轉(zhuǎn)化到大腸桿菌BL21(DE3)中經(jīng)IPTG誘導(dǎo)表達(dá);間接ELISA法檢驗(yàn)重組Bac蛋白特異性過(guò)敏原性;clustalw2進(jìn)行同源性分析,MEGA5工具包來(lái)構(gòu)建系統(tǒng)進(jìn)化樹(shù),ProtParam Tools預(yù)測(cè)其理化性質(zhì),PSIPRED預(yù)測(cè)其二級(jí)結(jié)構(gòu),SWISS-MODEL預(yù)測(cè)其三級(jí)結(jié)構(gòu)。利用網(wǎng)絡(luò)服務(wù)器IEDB內(nèi)相關(guān)軟件和Preprod,對(duì)Bac蛋白T細(xì)胞抗原表位進(jìn)行預(yù)測(cè)。用DNAStar對(duì)Bac的B細(xì)胞抗原表位進(jìn)行預(yù)測(cè)。結(jié)果經(jīng)測(cè)序鑒定,本研究成功克隆出了粉塵螨Bac基因,其開(kāi)放閱讀框396bp,編碼131組氨基酸。將Bac基因?qū)氪竽c桿菌E.coliBL21(DE3),經(jīng)IPTG誘導(dǎo)后高效表達(dá)Bac重組蛋白。該蛋白主要以可溶性形式存在,蛋白分子量約14kDa。間接ELISA法證明Bac能與粉塵螨過(guò)敏患者血清IgE結(jié)合。測(cè)序發(fā)現(xiàn)本實(shí)驗(yàn)室克隆的粉塵螨Bac基因與GenBank上公布的GenBank KF113885.1同源性為96%。系統(tǒng)進(jìn)化樹(shù)結(jié)果顯示粉塵螨與屋塵螨親緣關(guān)系比較近。理化性質(zhì)預(yù)測(cè)顯示Bac蛋白質(zhì)較穩(wěn)定。二級(jí)結(jié)構(gòu)及三級(jí)結(jié)構(gòu)預(yù)測(cè)結(jié)果顯示Bac的結(jié)構(gòu)主要以無(wú)規(guī)則卷曲組成。T細(xì)胞抗原表位預(yù)測(cè)得到4個(gè)肽序列(6-14、38-46、85-99、122-130)。B細(xì)胞抗原表位預(yù)測(cè)得到6個(gè)肽序列(15-30、26-40、44-59、58-73、95-110、101-116)。結(jié)論成功克隆并表達(dá)了粉塵螨Bac,并證實(shí)重組Bac蛋白具有良好的免疫原性,為進(jìn)一步研究塵螨過(guò)敏原的結(jié)構(gòu)成分及其理化性質(zhì)奠定理論基礎(chǔ)。
[Abstract]:Objective to clone and express the 13.8kDa lysozyme gene of Dermatophagoides farinae, purify and identify the allergen of the protein and analyze it by bioinformatics. Methods Total RNAs were extracted from pure cultured Dermatophagoides farinae and primers were designed according to known gene sequences. Bac gene fragments were amplified by RT-PCR and the products were linked into pMD-32T vector. After amplification, the target gene fragment was ligated to the expression vector of Pet-44a by restriction endonuclease EcoRI and XhoI digestion, and transformed into E. coli BL21DDE3) for expression induced by IPTG. Indirect ELISA assay to detect the specific allergen of recombinant Bac protein clustalw2 homology analysis was carried out to construct a phylogenetic tree named ProtParam Tools to predict its physical and chemical properties. PSIPRED predicted its secondary structure and SWISS-MODEL predicted its tertiary structure. The T cell epitopes of Bac protein were predicted by using the relative software of IEDB and preprod. B cell epitopes of Bac were predicted by DNAStar. Results the Bac gene of Dermatophagoides farinae was cloned successfully by sequencing, and its open reading frame was 396bp, encoding 131amino acids. The Bac gene was introduced into E. coli E. coli BL21 (DE3), and the recombinant Bac protein was highly expressed by IPTG induction. The protein mainly exists in soluble form and its molecular weight is about 14 kDa. Indirect ELISA assay demonstrated that Bac could bind to serum IgE in patients with Dermatophagoides farinae allergy. Sequencing showed that the cloned Bac gene of Dermatophagoides farinae in our laboratory had 96 homology with GenBank KF113885.1 published on GenBank. Phylogenetic tree results showed that the genetic relationship between Dermatophagoides farinae and housedust mites was close. The prediction of physicochemical properties showed that Bac protein was stable. The prediction results of secondary and tertiary structures showed that the structure of Bac was mainly predicted by irregular curl composition. T cell epitopes. Four peptide sequences 6-1438-46-45-99122-130. B cell epitopes were predicted and 6 peptide sequences were predicted, namely 15-30CG-4044-54-58-735-110101-116N. Conclusion Bacillus farinae was cloned and expressed successfully, and the recombinant Bac protein had good immunogenicity, which laid a theoretical foundation for further study on the structure and physical and chemical properties of dust mite allergen.
【作者單位】： 深圳大學(xué)過(guò)敏反應(yīng)和免疫學(xué)研究所;
【基金】：國(guó)家自然科學(xué)基金(No.91442118、No.31400786) 廣東省科技計(jì)劃社會(huì)發(fā)展項(xiàng)目(No.2013B03180002) 廣東省對(duì)外科技合作項(xiàng)目(No.2013B051000088) 廣東省公益研究與能力建設(shè)專項(xiàng)資金(No.2013B031800023) 深圳市科技計(jì)劃國(guó)際科技合作項(xiàng)目(No.GJHZ20130408174112021) 深圳市科技計(jì)劃基礎(chǔ)研究項(xiàng)目(No.JCYJ20140828163633992)聯(lián)合資助~~
【分類號(hào)】：R384.4

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