抗菌肽高效表达策略及活性研究
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摘要
利用基因工程手段生产抗菌肽的过程中,由于抗菌肽对宿主细胞会产生毒性使得蛋白表达受到影响,而改变融合头是使抗菌肽高效表达策略之一。本实验利用实验室已经成功构建并保存的工程菌,分析其诱导后OD600的变化以及不同融合头对G13毒性的影响。实验对pET28a-G13进行突变,以改变G13的N端融合头净正电荷数。设计突变引物,以质粒pET28a-G13为模板,PCR扩增序列,构建突变体pET28a'-G13,并进行诱导。对共表达工程菌在不同的抗生素比例下进行诱导,Tricine-SDS-PAGE电泳检测,比较分析抗生素比例对蛋白表达的影响,同时比较抗生素比例不同质粒的拷贝数的变化。试验发现融合头对G13毒性抑制效果与其净负电荷数及其氨基酸分布有关。
此外,针对本实验室已经摸索出的重组G13结构域的表达的方法进行改良,通过改变溶解蛋白的变性剂,以及改变其浓度等方法,希望能够减少尿素对重组肽的修饰。而且,在对纯化得到的G13进行了初步的检测之后,对于G13的活性研究进行了扩展,进一步研究G13结构域的溶血活性作用并对结果进行统计分析。
另外本实验对抗菌肽vgf-1的表达和活性进行了初步研究。通过对NCBI蛋白质数据库的检索查询找到了一个从眼镜蛇蛇毒中提取的具有抗结核杆菌效用的小肽Vgf-1。根据Vgf-1氨基酸序列和大肠杆菌的密码子偏好性,得出Vgf-1的基因序列。设计采用重叠PCR的方法得到基因序列。分三步扩增Vgf-1的基因片段(A,B,C),这三个片段经过胶回收后分别均与表达载体pBAD/TOPO连接,转化感受态细胞E.coli TOP 10,经过测序鉴定,分别命名为pBAD-Vgf-1-A,B,C。用阿拉伯糖诱导4h,经超声破碎处理后,SDS-PAGE电泳检测,融合蛋白的位置正确,且可以看出大多数目的蛋白以包涵体的形式存在。用Bradford方法测菌体总蛋白量,用Bandscan软件分析融合蛋白的表达量分别约占总体蛋白量的58.3%、48.7%和45.5%,因此推算出推算每升工程菌发酵液产生的包涵体约为178mg、142mg和124mg。对所得到的融合蛋白pBAD-Vgf-1-B,C进行肠激酶切割体系的优化,得出最佳的肠激酶切割体系。利用最佳体系对融合蛋白进行切割,经过活性检测发现,目的肽没有抑菌活性。实验尝试利用各种比例的GSH/GSSG复性体系对包涵体进行复性,浓缩后进行肠激酶切割,结果发现复性未能帮助得到有活性的蛋白。后采用先对融合蛋白进行切割,再进行复性的实验方案,试验结果表明仍为获得有活性的蛋白。
The toxicity of recombinant antimicrobial peptides upon the host cell affects the level of protein expression in genetic engineering. One of the strategies to achieve high expression is changing the fusion partners.
In this study, we analyze the OD600 of the engineering bacteria and the effects of different fusion partners on the inhibition of the toxicity of G13. We mutated the gene sequence of the fusion partners of pET28a-G13, the charge of the fusion partners of the mutant was changed in the N-terminal of G13. We designed the mutation primers. A PCR reaction was conducted with the primers and pET28a-G13 as template. The mutated recombinant named pET28a'-G13 and induced by IPTG. The double transformed cells are inoculated to different ratios of antibiotic, to examine the target protein protein expression level and the copy numbers of the plasmids. It was found that the inhibition effect of the fusion partners was greatly affected the net negative charge and the relative location of the acidic amino acid residues.
In addition, we tried to improve the method of the purification of G13 domain, to decrease the protein modification of urea through changing the denaturants and their concentrations. Moreover, we obtained purified G13 domain, tested its activity, further to study the hemolytic of it and do the conduct probability analysis.
Furthermore, we studied on the expression and activity of antimicrobial peptide vgf-1. We found an antimicrobial peptide (vgf-1) through the protein database of NCBI, that come from cobra venom and has effect on Mycobacterium tuberculosis. According to the protein sequence of vgf-1 and the codon preference of E. coli, we obtained the gene sequences of it. The gene sequence of Vgf-1 was obtained by overlapping PCR, which was amplified by three steps. The cleansed PCR product of three fragments were attach to the expression vector pBAD/TOPO, and transformed into E.coli TOP 10. After sequencing, we named them pBAD-Vgf-1-A, B, C. They were induced by arabinose, treated by ultrasonic treatment, the result of SDS-PAGE electrophoresis detection shows that fusion proteins were in correct position, and the target protein exists in the form of inclusion bodies. The total cell protein measured by Bradford, we analyze the the expression of fusion protein were about the overall protein content of 58.3%,48.7% and 45.5% with Bandscan software. So the projected per liter of fermentation broth produced works about inclusion as 178mg,142mg and 124mg.The enterokinase cutting system of fusion protein pBAD-Vgf-1-B, C was optimization. We used the best system to cut on the fusion protein, after activity detected the purpose of peptide has no antibacterial activity. We used various proportions of GSH/GSSG refolding system of the inclusion body, after concentrated and cutting, the protein has no activity. The other method was tried, after cutting the fusion protein, we treated the protein by the GSH/GSSG refolding system, the result showed that they has no activity.
此外,针对本实验室已经摸索出的重组G13结构域的表达的方法进行改良,通过改变溶解蛋白的变性剂,以及改变其浓度等方法,希望能够减少尿素对重组肽的修饰。而且,在对纯化得到的G13进行了初步的检测之后,对于G13的活性研究进行了扩展,进一步研究G13结构域的溶血活性作用并对结果进行统计分析。
另外本实验对抗菌肽vgf-1的表达和活性进行了初步研究。通过对NCBI蛋白质数据库的检索查询找到了一个从眼镜蛇蛇毒中提取的具有抗结核杆菌效用的小肽Vgf-1。根据Vgf-1氨基酸序列和大肠杆菌的密码子偏好性,得出Vgf-1的基因序列。设计采用重叠PCR的方法得到基因序列。分三步扩增Vgf-1的基因片段(A,B,C),这三个片段经过胶回收后分别均与表达载体pBAD/TOPO连接,转化感受态细胞E.coli TOP 10,经过测序鉴定,分别命名为pBAD-Vgf-1-A,B,C。用阿拉伯糖诱导4h,经超声破碎处理后,SDS-PAGE电泳检测,融合蛋白的位置正确,且可以看出大多数目的蛋白以包涵体的形式存在。用Bradford方法测菌体总蛋白量,用Bandscan软件分析融合蛋白的表达量分别约占总体蛋白量的58.3%、48.7%和45.5%,因此推算出推算每升工程菌发酵液产生的包涵体约为178mg、142mg和124mg。对所得到的融合蛋白pBAD-Vgf-1-B,C进行肠激酶切割体系的优化,得出最佳的肠激酶切割体系。利用最佳体系对融合蛋白进行切割,经过活性检测发现,目的肽没有抑菌活性。实验尝试利用各种比例的GSH/GSSG复性体系对包涵体进行复性,浓缩后进行肠激酶切割,结果发现复性未能帮助得到有活性的蛋白。后采用先对融合蛋白进行切割,再进行复性的实验方案,试验结果表明仍为获得有活性的蛋白。
The toxicity of recombinant antimicrobial peptides upon the host cell affects the level of protein expression in genetic engineering. One of the strategies to achieve high expression is changing the fusion partners.
In this study, we analyze the OD600 of the engineering bacteria and the effects of different fusion partners on the inhibition of the toxicity of G13. We mutated the gene sequence of the fusion partners of pET28a-G13, the charge of the fusion partners of the mutant was changed in the N-terminal of G13. We designed the mutation primers. A PCR reaction was conducted with the primers and pET28a-G13 as template. The mutated recombinant named pET28a'-G13 and induced by IPTG. The double transformed cells are inoculated to different ratios of antibiotic, to examine the target protein protein expression level and the copy numbers of the plasmids. It was found that the inhibition effect of the fusion partners was greatly affected the net negative charge and the relative location of the acidic amino acid residues.
In addition, we tried to improve the method of the purification of G13 domain, to decrease the protein modification of urea through changing the denaturants and their concentrations. Moreover, we obtained purified G13 domain, tested its activity, further to study the hemolytic of it and do the conduct probability analysis.
Furthermore, we studied on the expression and activity of antimicrobial peptide vgf-1. We found an antimicrobial peptide (vgf-1) through the protein database of NCBI, that come from cobra venom and has effect on Mycobacterium tuberculosis. According to the protein sequence of vgf-1 and the codon preference of E. coli, we obtained the gene sequences of it. The gene sequence of Vgf-1 was obtained by overlapping PCR, which was amplified by three steps. The cleansed PCR product of three fragments were attach to the expression vector pBAD/TOPO, and transformed into E.coli TOP 10. After sequencing, we named them pBAD-Vgf-1-A, B, C. They were induced by arabinose, treated by ultrasonic treatment, the result of SDS-PAGE electrophoresis detection shows that fusion proteins were in correct position, and the target protein exists in the form of inclusion bodies. The total cell protein measured by Bradford, we analyze the the expression of fusion protein were about the overall protein content of 58.3%,48.7% and 45.5% with Bandscan software. So the projected per liter of fermentation broth produced works about inclusion as 178mg,142mg and 124mg.The enterokinase cutting system of fusion protein pBAD-Vgf-1-B, C was optimization. We used the best system to cut on the fusion protein, after activity detected the purpose of peptide has no antibacterial activity. We used various proportions of GSH/GSSG refolding system of the inclusion body, after concentrated and cutting, the protein has no activity. The other method was tried, after cutting the fusion protein, we treated the protein by the GSH/GSSG refolding system, the result showed that they has no activity.
引文
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