抗菌肽SMAP-29基因的設(shè)計、合成、表達純化及抗菌活性的檢測
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【摘要】：目的： 通過設(shè)計合成抗菌肽SMAP-29新基因，構(gòu)建其原核表達載體pGEX-4T-1／SMAP-29，經(jīng)誘導(dǎo)表達和分離純化，得到大量具有抗菌活性的抗菌肽SMAP-29。 方法： 根據(jù)SMAP-29蛋白質(zhì)氨基酸序列，選用大腸桿菌偏愛密碼子，同時兼顧了其mRNA二級結(jié)構(gòu)自由能自行設(shè)計全新基因，基因采用geneSOEing(gene splicing by overlap extension，geneSOEing)法合成，合成的基因克隆至pGEX-4T-1原核表達載體中，通過酶切分析、PCR和測序鑒定篩選出陽性重組質(zhì)粒；重組質(zhì)粒轉(zhuǎn)化到大腸桿菌BL21(DE3)中進行IPTG誘導(dǎo)表達，經(jīng)GST瓊脂糖FF層析柱純化，同時作凝血酶切割，再經(jīng)HPLC分離純化，獲得純度達95％的目的蛋白。檢測抗菌肽SMAP—29的抗菌活性，采用微量肉湯稀釋法測定其對白色葡萄球菌(臨床株)、糞腸球菌耐藥株(耐氟喹諾酮類)、糞腸球菌標(biāo)準(zhǔn)株(ATCC29212)和大腸桿菌(JM 109)的MIC和MBC值。 結(jié)果： 經(jīng)geneSOEing法合成的全新基因長109bp(含限制性酶切位點)，將其克隆至pGEX-4T-1原核表達載體中，通過酶切分析、PCR和測序鑒定，其序列與所設(shè)計序列完全一致。SDS—PAGE電泳結(jié)果分析表明pGEX-4T-1／SMAP-29可以在BL21中表達大小約29KD的GST-SMAP-29融合蛋白，經(jīng)凝血酶切割和分離純化后得到大小約3.2KDa的SMAP-29目的多肽。微量肉湯稀釋法檢測表明SMAP-29具有較強的抗菌活性。
[Abstract]:Objective: to construct the prokaryotic expression vector pGEX-4T-1/SMAP-29, by designing and synthesizing a novel gene of antimicrobial peptide SMAP-29. A large number of antimicrobial peptides SMAP-29. with antimicrobial activity were obtained. Methods: according to the amino acid sequence of SMAP-29 protein, Escherichia coli preference codon was selected and its mRNA secondary structure free energy was used to design a new gene. The gene was synthesized by geneSOEing (gene splicing by overlap extension,geneSOEing and cloned into the prokaryotic expression vector of pGEX-4T-1. The positive recombinant plasmid was screened by restriction endonuclease analysis, PCR and sequencing. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for IPTG induction expression, purified by GST agarose FF chromatography column, then digested by thrombin, then purified by HPLC, and the purity of target protein was 95%. The antimicrobial activity of antimicrobial peptide SMAP-29 was determined by broth dilution method. The MIC and MBC values of the antimicrobial peptide SMAP-29 against Staphylococcus albus (clinical strain), Enterococcus faecalis resistant strain (fluoroquinolone resistance), Enterococcus faecalis standard strain (ATCC29212) and Escherichia coli (JM 109) were determined. Results: the new gene long 109bp (including restriction enzyme site) synthesized by geneSOEing method was cloned into pGEX-4T-1 prokaryotic expression vector and identified by enzyme digestion, PCR and sequencing. The results of SDS-PAGE electrophoresis showed that pGEX-4T-1/SMAP-29 could express GST-SMAP-29 fusion protein about the size of 29KD in BL21. After thrombin cleavage and purification, the target polypeptide of SMAP-29 of about 3.2KDa size could be obtained. Trace broth dilution assay showed that SMAP-29 had strong antibacterial activity.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346
本文編號：2276580
[Abstract]:Objective: to construct the prokaryotic expression vector pGEX-4T-1/SMAP-29, by designing and synthesizing a novel gene of antimicrobial peptide SMAP-29. A large number of antimicrobial peptides SMAP-29. with antimicrobial activity were obtained. Methods: according to the amino acid sequence of SMAP-29 protein, Escherichia coli preference codon was selected and its mRNA secondary structure free energy was used to design a new gene. The gene was synthesized by geneSOEing (gene splicing by overlap extension,geneSOEing and cloned into the prokaryotic expression vector of pGEX-4T-1. The positive recombinant plasmid was screened by restriction endonuclease analysis, PCR and sequencing. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for IPTG induction expression, purified by GST agarose FF chromatography column, then digested by thrombin, then purified by HPLC, and the purity of target protein was 95%. The antimicrobial activity of antimicrobial peptide SMAP-29 was determined by broth dilution method. The MIC and MBC values of the antimicrobial peptide SMAP-29 against Staphylococcus albus (clinical strain), Enterococcus faecalis resistant strain (fluoroquinolone resistance), Enterococcus faecalis standard strain (ATCC29212) and Escherichia coli (JM 109) were determined. Results: the new gene long 109bp (including restriction enzyme site) synthesized by geneSOEing method was cloned into pGEX-4T-1 prokaryotic expression vector and identified by enzyme digestion, PCR and sequencing. The results of SDS-PAGE electrophoresis showed that pGEX-4T-1/SMAP-29 could express GST-SMAP-29 fusion protein about the size of 29KD in BL21. After thrombin cleavage and purification, the target polypeptide of SMAP-29 of about 3.2KDa size could be obtained. Trace broth dilution assay showed that SMAP-29 had strong antibacterial activity.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346
【引證文獻】
相關(guān)期刊論文 前1條
1 任耀軍;王新華;薄新文;;抗菌肽SMAP-29研究進展[J];中國預(yù)防獸醫(yī)學(xué)報;2008年11期
，本文編號：2276580
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