抗菌肽ABPS1提取纯化的初步研究及其与干扰素IFNa-2b融合表达载体的构建
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摘要
抗菌肽(antibacterial peptide, ABP)是一种具有广谱抗菌活性的小肽类物质。研究表明,除了抗菌活性外,抗菌肽还具有抗病毒、抗真菌和抗肿瘤作用。干扰素(interferon, IFN)是一类具有广谱抗病毒活性的糖蛋白,而且还有抑制肿瘤增殖、抗纤维化以及免疫调节等功能。
在已有高效表达融合蛋白ABPS1-GFP的基因工程菌的基础上,对融合蛋白的纯化条件进行了初步研究。首先将高效表达抗菌肽ABPS1的工程菌扩大培养,诱导表达后所得的菌体离心后进行冰浴下超声裂解,继而用表面活性剂TritonX-100和低浓度的尿素对包涵体进行充分洗涤,接着用高浓度的尿素裂解包涵体,然后在变性条件下对融合蛋白进行Ni-NTA亲和层析纯化,最后采用尿素梯度透析法对变性蛋白进行复性,并对包涵体变性、复性等条件进行了初步优化。
然而,将融合蛋白ABPS1-GFP裂解获得抗菌肽ABPS1的蛋白单体,必须使用化学裂解剂CNBr。CNBr是一种剧毒化学物质,很难购买;而且在基因工程产品的生产中,禁止使用CNBr。为此,我们对此表达载体进行了改造,构建了含有1个IFNa-2b基因和2个ABPS1基因的融合表达载体。首先在基因水平上,通过PCR的方法在各单体分子之间引入凝血因子Xa切割位点的识别序列,以质粒pET-28b(+)为载体,利用同裂酶BamHI与BglⅡ可以切出相同粘性末端的特性,通过酶切、连接、转化等操作,构建干扰素和抗菌肽的融合表达载体。经PCR、双酶切和DNA测序分析表明,串联体的DNA序列及阅读框完全正确。将干扰素IFNα-2b基因和抗菌肽ABPS1基因融合在一起,在大肠杆菌中进行表达,通过纯化后裂解可以同时获得这两种蛋白,为将ABPS1和IFNα-2b单独或联合应用于临床和生产实践奠定基础。
The antibacterial peptide (ABPS1) is a small peptide which has a wide range of biological activities such as antimicrobial activities, antivirus and antitumor activities. And interferon is a key member of the glycosylated cytokines that share the capacity to inhibit viral replication and to exert effects on immune function in the host immunity. In this study, in order to obtain the fusion protein of IFNα -2b and ABPS1, the co-expression vector of IFNα -2b and ABPS1 genes was constructed and expressed in E.coli.On the base of high expression of ABPS1-GFP fusion protein in E.coli, the purification of expression product was performed. The fusion protein was highly expressed in the form of inclusion body. The bodies of engineering E. coli were harvested by centrifugation and disturbed by sonication, then inclusion body was extracted and washed with buffer consisting of urea and TritonX-100 for several times. The inclusion body was dissolved in the denature buffer using urea as denaturant, and then purified under urea denature condition using nickel chelate affinity chromatography method. A higher purity of the denatured inclusion body protein was attained. After renaturation through gradient dialysis method, a ideal renatured protein was obtained.However, there exists CNBr which is a kind of toxin and is difficult to buy. Furthermore, it is forbidden in production of gene engineering product.So we constructed Prokaryotic Fusion Expression Vector of ABPS1 and Interferon a-2b.The antibacterial peptide Shiva-1 gene and Interferon a-2b gene were subcloned by PCR respectively and inserted into pET-28b(+) expression plasmid in turn by restriction endonuclease.Then the multimer was constructed by a pair of isoschizomers, BamHI and Bgl II, which could produce the same cohesive end (GATC) when cut its target DNA sequence, their digested fragments could be ligated by T4 ligase and could not be excised by Bgl II or BamHI again. The recombinant plasmids containing IFN-a-2b and two copies of ABPS1 gene was transformed into Escherichia coli BL21 (DE3) plysS and then induced by IPTG to express fusion protein. The multimers of IFN α -2b and ABPS1 has been successfully constructed by restriction endonuclease and direct DNA sequencing. This benefits preparation of IFNα -2b and ABPS1 in a large scale and provides an experimental data for the production of other small bioactive peptides.
在已有高效表达融合蛋白ABPS1-GFP的基因工程菌的基础上,对融合蛋白的纯化条件进行了初步研究。首先将高效表达抗菌肽ABPS1的工程菌扩大培养,诱导表达后所得的菌体离心后进行冰浴下超声裂解,继而用表面活性剂TritonX-100和低浓度的尿素对包涵体进行充分洗涤,接着用高浓度的尿素裂解包涵体,然后在变性条件下对融合蛋白进行Ni-NTA亲和层析纯化,最后采用尿素梯度透析法对变性蛋白进行复性,并对包涵体变性、复性等条件进行了初步优化。
然而,将融合蛋白ABPS1-GFP裂解获得抗菌肽ABPS1的蛋白单体,必须使用化学裂解剂CNBr。CNBr是一种剧毒化学物质,很难购买;而且在基因工程产品的生产中,禁止使用CNBr。为此,我们对此表达载体进行了改造,构建了含有1个IFNa-2b基因和2个ABPS1基因的融合表达载体。首先在基因水平上,通过PCR的方法在各单体分子之间引入凝血因子Xa切割位点的识别序列,以质粒pET-28b(+)为载体,利用同裂酶BamHI与BglⅡ可以切出相同粘性末端的特性,通过酶切、连接、转化等操作,构建干扰素和抗菌肽的融合表达载体。经PCR、双酶切和DNA测序分析表明,串联体的DNA序列及阅读框完全正确。将干扰素IFNα-2b基因和抗菌肽ABPS1基因融合在一起,在大肠杆菌中进行表达,通过纯化后裂解可以同时获得这两种蛋白,为将ABPS1和IFNα-2b单独或联合应用于临床和生产实践奠定基础。
The antibacterial peptide (ABPS1) is a small peptide which has a wide range of biological activities such as antimicrobial activities, antivirus and antitumor activities. And interferon is a key member of the glycosylated cytokines that share the capacity to inhibit viral replication and to exert effects on immune function in the host immunity. In this study, in order to obtain the fusion protein of IFNα -2b and ABPS1, the co-expression vector of IFNα -2b and ABPS1 genes was constructed and expressed in E.coli.On the base of high expression of ABPS1-GFP fusion protein in E.coli, the purification of expression product was performed. The fusion protein was highly expressed in the form of inclusion body. The bodies of engineering E. coli were harvested by centrifugation and disturbed by sonication, then inclusion body was extracted and washed with buffer consisting of urea and TritonX-100 for several times. The inclusion body was dissolved in the denature buffer using urea as denaturant, and then purified under urea denature condition using nickel chelate affinity chromatography method. A higher purity of the denatured inclusion body protein was attained. After renaturation through gradient dialysis method, a ideal renatured protein was obtained.However, there exists CNBr which is a kind of toxin and is difficult to buy. Furthermore, it is forbidden in production of gene engineering product.So we constructed Prokaryotic Fusion Expression Vector of ABPS1 and Interferon a-2b.The antibacterial peptide Shiva-1 gene and Interferon a-2b gene were subcloned by PCR respectively and inserted into pET-28b(+) expression plasmid in turn by restriction endonuclease.Then the multimer was constructed by a pair of isoschizomers, BamHI and Bgl II, which could produce the same cohesive end (GATC) when cut its target DNA sequence, their digested fragments could be ligated by T4 ligase and could not be excised by Bgl II or BamHI again. The recombinant plasmids containing IFN-a-2b and two copies of ABPS1 gene was transformed into Escherichia coli BL21 (DE3) plysS and then induced by IPTG to express fusion protein. The multimers of IFN α -2b and ABPS1 has been successfully constructed by restriction endonuclease and direct DNA sequencing. This benefits preparation of IFNα -2b and ABPS1 in a large scale and provides an experimental data for the production of other small bioactive peptides.
引文
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