抗菌肽SMAP-29基因的设计、合成、表达纯化及抗菌活性的检测
发布时间：2018-10-17 11:57
【摘要】：目的： 通过设计合成抗菌肽SMAP-29新基因，构建其原核表达载体pGEX-4T-1／SMAP-29，经诱导表达和分离纯化，得到大量具有抗菌活性的抗菌肽SMAP-29。 方法： 根据SMAP-29蛋白质氨基酸序列，选用大肠杆菌偏爱密码子，同时兼顾了其mRNA二级结构自由能自行设计全新基因，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原核表达载体中，通过酶切分析、PCR和测序鉴定筛选出阳性重组质粒；重组质粒转化到大肠杆菌BL21(DE3)中进行IPTG诱导表达，经GST琼脂糖FF层析柱纯化，同时作凝血酶切割，再经HPLC分离纯化，获得纯度达95％的目的蛋白。检测抗菌肽SMAP—29的抗菌活性，采用微量肉汤稀释法测定其对白色葡萄球菌(临床株)、粪肠球菌耐药株(耐氟喹诺酮类)、粪肠球菌标准株(ATCC29212)和大肠杆菌(JM 109)的MIC和MBC值。 结果： 经geneSOEing法合成的全新基因长109bp(含限制性酶切位点)，将其克隆至pGEX-4T-1原核表达载体中，通过酶切分析、PCR和测序鉴定，其序列与所设计序列完全一致。SDS—PAGE电泳结果分析表明pGEX-4T-1／SMAP-29可以在BL21中表达大小约29KD的GST-SMAP-29融合蛋白，经凝血酶切割和分离纯化后得到大小约3.2KDa的SMAP-29目的多肽。微量肉汤稀释法检测表明SMAP-29具有较强的抗菌活性。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
本文编号：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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
【引证文献】
相关期刊论文 前1条
1 任耀军;王新华;薄新文;;抗菌肽SMAP-29研究进展[J];中国预防兽医学报;2008年11期
，本文编号：2276580
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2276580.html
最近更新
教材专著
