本文選題：疥螨 + 絲切蛋白��； 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：疥螨病作為一種重要的人獸共患寄生蟲病,嚴(yán)重影響人類健康和生活質(zhì)量,特別是在人口密度較大的區(qū)域內(nèi)流行廣泛,另外還能危害多種動物,給社會經(jīng)濟(jì)帶來不可小覷的損失。目前該病已引起了世界許多國家和組織機(jī)構(gòu)的重視,針對疥螨的分子學(xué)研究不斷深入,但還沒有有效的免疫學(xué)診斷方法和疫苗問世。本研究根據(jù)NCBI數(shù)據(jù)庫中疥螨絲切蛋白基因的EST序列設(shè)計(jì)引物,通過擴(kuò)增、克隆后獲得447bp大小的目的基因片段,將測序結(jié)果在GenBamk數(shù)據(jù)庫中比對后發(fā)現(xiàn)與已報(bào)道的塵螨主要變應(yīng)原之一的Derf31基因(GenBank:KM010014.1)的序列相似性最高(90%)。根據(jù)軟件預(yù)測該絲切蛋白分子量約為16.8kDa,等電點(diǎn)為pI=5.59,推導(dǎo)化學(xué)式為C751H1175N1910231S8,蛋白帶負(fù)電荷,無信號肽切割位點(diǎn),不存在跨膜結(jié)構(gòu)域,是一種膜外蛋白。將目的基因片段連入表達(dá)載體pET32a(+)后轉(zhuǎn)入表達(dá)菌大腸桿菌E.coliBL21(DE3),并優(yōu)化誘導(dǎo)條件,通過聚丙烯凝膠電泳驗(yàn)證該蛋白表達(dá)后,利用Ni-親和層析柱純化疥螨絲切蛋白。在免疫印跡實(shí)驗(yàn)中,疥螨絲切蛋白重組蛋白與兔疥螨陽性血清反應(yīng)后產(chǎn)生免疫印跡條帶,說明其具有良好的抗原性。利用純化后的疥螨絲切重組蛋白免疫制備疫苗免疫實(shí)驗(yàn)兔,將采集到的高免血清作為一抗用于熒光組織化學(xué)實(shí)驗(yàn),結(jié)果發(fā)現(xiàn)該蛋白廣泛分布于疥螨蟲體組織中,但表皮中未檢測到該蛋白存在。將純化后的絲切重組蛋白作抗原,經(jīng)過條件優(yōu)化篩選和特異性、重復(fù)性試驗(yàn),成功建立了間接ELISA檢測方法。實(shí)驗(yàn)結(jié)果確定抗原最佳包被濃度為5μg/mL,血清的最佳工作濃度為1:100,酶標(biāo)二抗最適使用濃度為1:3000。利用cut-off計(jì)算公式確定陰陽性臨界值,最后得到cut-off值為0.188,即當(dāng)血清OD450≥ 0.188判為陽性,反之為陰性。再根據(jù)公式計(jì)算出特異性87.9%,敏感性83.33%。另外該方法檢測出,該重組蛋白與患癢螨的和患豆?fàn)钅椅豺实耐藐栃匝寰话l(fā)生交叉反應(yīng);批內(nèi)變異系數(shù)在1.28%～4.08%之間,批間變異系數(shù)為1.81%～5.30%之間。本試驗(yàn)證明疥螨絲切重組蛋白具有潛在的診斷價值。
[Abstract]:As an important zoonotic parasitic disease, scabies seriously affects human health and the quality of life, especially in areas with high population density.To the social economy brings the loss which cannot be underestimated.At present, the disease has attracted the attention of many countries and organizations in the world. Molecular studies on scabies mite have been deepened, but there is no effective immunological diagnosis method and vaccine.In this study, primers were designed according to the EST sequence of the filamentous protein gene of scabies mite in NCBI database, and the target gene fragment of 447bp size was obtained by amplification and cloning.The sequencing results were compared in GenBamk database and the sequence similarity was the highest with the reported Derf31 gene GenBank: KM010014.1).According to the software prediction, the molecular weight of the protein is about 16.8 kDa and the isoelectric point is Pi 5.59.The deduced chemical formula is C751H1175N1910231S8. The protein has negative charge, no signal peptide cleavage site, no transmembrane domain, and is an extramembrane protein.The target gene fragment was inserted into the expression vector pET32a () and then transferred into E. coli BL21DE3, and the induction conditions were optimized. The protein was identified by polyacrylamide gel electrophoresis and purified by Ni-affinity chromatography.In the Western blotting experiment, the recombinant protein of scabies mites silk cut protein reacted with the positive serum of the rabbit scabies mite to produce the immunoblotting bands, which indicated that the recombinant protein had good antigenicity.The immunized rabbits were immunized with purified recombinant protein of scabies mites. The high immune serum was used as a first antibody for fluorescent histochemical experiments. It was found that the protein was widely distributed in the tissue of scabies mite.However, the protein was not detected in the epidermis.The purified recombinant protein was used as antigen, and the indirect ELISA detection method was successfully established by optimized screening, specificity and repeatability test.The results showed that the best coating concentration of antigen was 5 渭 g / mL, the optimal working concentration of serum was 1: 100, and the optimum concentration of enzyme labeled second antibody was 1: 3000.The critical value of yin and yang was determined by using the cut-off formula, and the final cut-off value was 0.1888, that is, when serum OD450 鈮，

本文編號：1754754