本文選題：鉤端螺旋體 + 轉(zhuǎn)座�。� 參考：《沈陽藥科大學(xué)》2005年碩士論文

【摘要】：鉤端螺旋體中國有毒賴株的全基因組測序已經(jīng)完成，這只是鉤端螺旋體功能基因組研究的第一步。在中國有毒賴株中共有大小兩個染色體，共4691184對核苷酸，預(yù)知含有4727個功能基因。大量基因的功能需要我們通過實(shí)驗(yàn)的方法來推知和證明。將未知功能的基因組核苷酸序列轉(zhuǎn)化為有意義的基因功能信息將是一個具有挑戰(zhàn)性的工作。 本實(shí)驗(yàn)獨(dú)立進(jìn)行了鉤端螺旋體無毒賴株和有毒賴株培養(yǎng)條件的研究。同時考察了甘氨酸，Mg~(2+)，DMSO，MTT，，溫度等對鉤體生長的影響，掌握了鉤體各種培養(yǎng)條件。用鉤端螺旋體無毒賴株作為研究對象摸索了鉤體的電轉(zhuǎn)化條件，研究了鉤端螺旋體有毒賴株的抗藥性，并建立了一套有效的抗性篩選方法，為鉤端螺旋體有毒賴株的全基因組突變體庫的建立創(chuàng)建了平臺。 應(yīng)用轉(zhuǎn)座子對微生物基因的插入突變是研究微生物全基因組功能的常用方法和有力手段，被廣泛應(yīng)用到模式生物的功能基因組學(xué)研究當(dāng)中。通過單引物PCR可以將插入位點(diǎn)在基因組中定位，通過表型的分析可以探知突變基因在基因組中的生物學(xué)功能。本實(shí)驗(yàn)通過Tn5體轉(zhuǎn)座系統(tǒng)成功摸索出了鉤端螺旋體無毒賴株的突變方法，并將Tn5體轉(zhuǎn)座系統(tǒng)加以修飾應(yīng)用于鉤端螺旋體有毒賴株的突變體庫的建立當(dāng)中去。在今后的工作中繼續(xù)構(gòu)建整合有帶有鉤體啟動子的轉(zhuǎn)座酶以及多轉(zhuǎn)座位點(diǎn)的體內(nèi)轉(zhuǎn)座體系。 本實(shí)驗(yàn)同時構(gòu)建了大腸桿菌ET12567接合轉(zhuǎn)移系統(tǒng)，與鉤體有毒賴株共培養(yǎng)，以帶有鉤體鞭毛蛋白基因flgH和抗性篩選標(biāo)記的接合載體來摸索鉤體單基因的插入失活和基因敲除方法。 本實(shí)驗(yàn)對鉤體中的必須基因金屬酶蛋白基因進(jìn)行了研究，并以肽鍵去甲基化酶(PDF)蛋白為目標(biāo)，應(yīng)用dock分子對接軟件搜索specs化合物數(shù)據(jù)庫，應(yīng)用體外抗菌實(shí)驗(yàn)的方法篩選基于PDF藥物靶點(diǎn)的抗菌小分子有機(jī)化合物，并申請新的化合物專利十項(xiàng)。
[Abstract]:The complete genome sequencing of Leptospira chinensis is the first step in the study of Leptospira functional genome.A total of 4691184 pairs of nucleotides were found in two chromosomes of Chinese virulent Lai strain, and 4727 functional genes were predicted.The function of a large number of genes needs to be deduced and proved by experimental methods.It will be a challenging task to transform unknown functional nucleotide sequences into meaningful gene functional information.The culture conditions of Leptospira non-venomous and venomous Leptospira were studied in this experiment.The effects of temperature and temperature on the growth of Leptospira were investigated, and various culture conditions of Leptospira were studied.The electrotransformation conditions of leptospirosis were studied by using leptospira nontoxic Lai strain as the research object. The resistance of leptospira venomous Lai strain was studied, and a set of effective screening methods were established.A platform was established for the establishment of a whole genome mutant library of Leptospira venomous Leptospira.The insertion mutation of microbial gene by transposon has been widely used in the functional genomics of model organisms.The insertion site can be located in the genome by single primer PCR, and the biological function of the mutant gene in the genome can be found by phenotypic analysis.In this experiment, the mutation method of leptospira venomous rogue strain was successfully explored by Tn5 transposition system, and the Tn5 transposition system was modified and applied to the establishment of the mutant library of Leptospira venomous Leptospira.In the future, we will continue to construct an in vivo transposable system with Leptospira promoter and multiple transposable loci.At the same time, we constructed E. coli ET12567 conjugation transfer system, co-cultured with Leptospira venomous Leptospira strain, and explored the method of insertion inactivation and gene knockout of Leptospira single gene with Leptospira flagellin gene flgH and resistance screening marker.In this study, the essential gene metalloproteinase gene in Leptospira was studied, and the specs compound database was searched by using dock molecular docking software with peptide desmethylase (PDF) protein as the target.Antimicrobial small molecule organic compounds based on PDF drug target were screened by in vitro antibacterial experiment and ten new compounds were patented.
【學(xué)位授予單位】：沈陽藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R377

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 夏英武,吳殿星,舒慶堯;農(nóng)桿菌T—DNA介導(dǎo)的植物轉(zhuǎn)基因的分子機(jī)制[J];生物學(xué)雜志;1994年05期

本文編號：1771205