奶牛子宫内膜抗菌肽分离纯化、BNBD5基因的克隆及原核表达
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摘要
内源性抗微生物肽是生物体内先天性免疫的重要组成部分,防御素是广泛分布于动物和植物界的一类富含半胱氨酸的阳离子内源性抗微生物肽,是内源性抗微生物肽中的一个大家族。是由生物细胞特定基因编码,经特定外界条件诱导产生的一类多肽。具有抵抗外界微生物侵害、消除体内突变细胞的作用。根据防御素分子内半胱氨酸的位置和连接方式、前体性质及表达位置的差异可分为α-防御素、β-防御素、θ-防御素、昆虫防御素和植物防御素五种类型,防御素主要分布于哺乳动物的黏膜上皮细胞内,所以,防御素被确认为黏膜表面抗微生物屏障的组成成分。
在实验中以奶牛子宫为实验材料,从奶牛子宫内膜上皮组织中提取总RNA,根据已发表的牛抗菌肽基因序列设计引物。经RT-PCR扩增抗菌肽DNA片段,回收纯化PCR产物,连接pMD18-T载体,转化感受态细胞DH5a。在含X-Gal、Amp及IPTG的LB平板上筛选阳性克隆,经PCR及双酶切鉴定后,对目的片段进行测序。结果表明,RT-PCR扩增出的cDNA片段碱基数为138bp,编码45个氨基酸的多肽,多肽中含6个保守半胱氨酸残基。用Blast程序进行检索比较,表明扩增序列与GenBank中发表的BNBD5编码区序列99.3%相同。利用DNAStar MegAlign分析了奶牛子宫内膜抗菌肽BNBD5与其他物种β-防御素5的ORF序列,得出奶牛BNBD5的cDNA与其他动物的β-防御素5的同源性。与山羊(Capra hircus,DQ532360)有80.4%的同源性,与小鼠(Mus musculus, NM_030734)有56.5%的同源性,与褐鼠(Rattus norvegicus,NM_001037549)有31.9%的同源性,而与冈比亚按蚊(Anopheles gambiae,EU273600)、中华蜜蜂(Apis cerana cerana,EU727272)和原鸡(Gallus gallus,AY621320、AY621307、DQ677636)的同源性均低于20%。这说明反刍动物的β防御素cDNA具有种属特异性。
再以重组质粒为模板,用特异性表达引物扩增得到的基因:即编码BNBD5基因全长cDNA连接到原核表达载体pET28a中,构建的原核表达质粒pET- BNBD5,转化大肠杆菌BL21(DE3)。结果表明:成功构建了pET28a-BNBD5载体,经IPTG的诱导, pET28a-BNBD5在原核内表达产物相对分子质量约为10ku,与预期融合蛋白大小一致,并以包涵体的形式存在,琼脂扩散实验检测表达产物的体外抑菌活性,在大肠杆菌中表达的奶牛子宫内膜抗菌肽的体外抑菌活性不太明显。
本实验再以奶牛子宫为实验材料,刮取内膜上皮,用5%的冰乙酸抽提,通过酸性尿素聚丙烯酰胺电泳洗脱技术和反相高效液相色谱技术,分离纯化奶牛子宫黏液内源性抗菌肽,并进行其抗菌活性的检测。活性检测结果表明:在5min收集的样品没有抑菌圈出现,在45min收集的样品具有抗大肠杆菌BL21和金黄色葡萄球菌26003的活性。
奶牛子宫内膜防御素的发现有利于对奶牛黏膜的防御机制进行进一步的研究,从而为奶牛子宫内膜炎防治奠定了基础。
Endogenous antimicrobial peptides are an important component in innate immunity of organism defensins are a kind of positive Endogenous antimicrobial peptides, which contain cysteine-rich and distribute abroad in kingdom of animals and plants. Defensins are a large family among cationic endogenous antimicrobial peptide. As a kind of polypeptide,controlled by specific genic code and induced under specific external conditions.Endogenous antimicrobial peptides can resist the invasion of outside microorganism and kill inner mutant cells . According to the differences of position and connect manner of cysteines,the property of precursors,the position of expression, defensins are divided into five types:α-defensins,β-defensins,θ-defensins, insect defensins and plant defensins,β-defensins are mainly proved to be a antimicrobial barrier bacause they distribute in mucosal epithelial cells of mammals and aves.
In this study ,we have used cow as experiment. As a pair of primers were designed and synthesized according to the sequence of antibacterial peptide gene published in GenBank. Total RNA was purified from the bovine endometrial cells with the total RNA isolation kit. Then the DNA fragment of bovine antibacterial peptide was amplified by RT-PCR. After gel purification,the RT–PCR product was cloned into a pMD18-T Vector System. The vector mixtures were transformed into competent high DH5a. Some positive clones were selected on the LB agar-plate with X-Gal,Amp and IPTG. Length of the inserted DNA fragment was confirmed using BamHⅠand HindⅢdigestion and PCR, then was sequenced. The result showed that the obtained 138bp DNA fragment is identical to the BNBD5 sequence registered in the GenBank. The cDNA fragment code 45 amino acid,which contained six invariantly aminothiopropionic acid residues. Compared to bovine antibacterial peptide,it shared the highest identity of 99.3% with the BNBD5.We analysised ORF sequences of the Endometrial cows and other species BNBD5 antimicrobial peptidesβ-defensin-5 by DNAStar MegAlign of computer sofeware, It indicated that the relationship of cow to Capra hircus(DQ532360) is the highest (80.4%),to theβ-defensins of Mus musculus (NM_030734))and Rattus norvegicus (NM_001037549)is middle(56.5%,31.9%),to theβ-defensins of Anopheles gambiae (EU273600),Apis cerana cerana(EU727272) and Gallus gallus (AY621320, AY621307, DQ677636) is the lowest (mostly less than 20%), This shows thatβ-defensins of ruminants are species-specific.
And then target genes ,that was fragments of cow which coded the BNBD5 (138bp), were amplified by PCR with a pair of specifically expessive primers and recombinant plasmid as a template respectively, then cloned into pET28a expession vector. We Constructed prokaryotic expression plasmid pET-BNBD5 and transformed into Escherichia coli BL21.Established highly expression target protein inducing by IPTG. Protein molecular weight is about 10 KDa. in coli BL21 with the same size of expection . the best conditions of inducting were detemined that the induction time was six hours,the temperature was 37℃,and the IPTG Concentration was0.5mM. Under these conditions,the expressed recombinant protein existed in the form of inclusion bodies. By detecting the expressed recombinant protein with the method of agar geldiffusion test,theβ-defensins from the endometrial epithelial of cow , expressed in E. coli ,showed that antibacterial activity is not obvious in vitro.
The cow was involved in the experiment. Endometrial epithelial was isolated and acid soluble extract of cow was obtained and antibacterial activity was detected by gel overlay technique , Then it was further isolated and purified by HPLC and analyzed antibacterial activity by agarose radial diffusion assay.The molecular weight was determined by Tricine-SDS-PAGE. The result showed that it was able to effectively kill E. coliBL21 and the Staphylococcus bacteria 26003, its molecular weight was about 14KDa.
The discovery of BNBD5 of Endometrial of cow provides evidence for understanding the mucosal defense mechanism and laid the foundation of preventing and curing the endometritis of cow .
在实验中以奶牛子宫为实验材料,从奶牛子宫内膜上皮组织中提取总RNA,根据已发表的牛抗菌肽基因序列设计引物。经RT-PCR扩增抗菌肽DNA片段,回收纯化PCR产物,连接pMD18-T载体,转化感受态细胞DH5a。在含X-Gal、Amp及IPTG的LB平板上筛选阳性克隆,经PCR及双酶切鉴定后,对目的片段进行测序。结果表明,RT-PCR扩增出的cDNA片段碱基数为138bp,编码45个氨基酸的多肽,多肽中含6个保守半胱氨酸残基。用Blast程序进行检索比较,表明扩增序列与GenBank中发表的BNBD5编码区序列99.3%相同。利用DNAStar MegAlign分析了奶牛子宫内膜抗菌肽BNBD5与其他物种β-防御素5的ORF序列,得出奶牛BNBD5的cDNA与其他动物的β-防御素5的同源性。与山羊(Capra hircus,DQ532360)有80.4%的同源性,与小鼠(Mus musculus, NM_030734)有56.5%的同源性,与褐鼠(Rattus norvegicus,NM_001037549)有31.9%的同源性,而与冈比亚按蚊(Anopheles gambiae,EU273600)、中华蜜蜂(Apis cerana cerana,EU727272)和原鸡(Gallus gallus,AY621320、AY621307、DQ677636)的同源性均低于20%。这说明反刍动物的β防御素cDNA具有种属特异性。
再以重组质粒为模板,用特异性表达引物扩增得到的基因:即编码BNBD5基因全长cDNA连接到原核表达载体pET28a中,构建的原核表达质粒pET- BNBD5,转化大肠杆菌BL21(DE3)。结果表明:成功构建了pET28a-BNBD5载体,经IPTG的诱导, pET28a-BNBD5在原核内表达产物相对分子质量约为10ku,与预期融合蛋白大小一致,并以包涵体的形式存在,琼脂扩散实验检测表达产物的体外抑菌活性,在大肠杆菌中表达的奶牛子宫内膜抗菌肽的体外抑菌活性不太明显。
本实验再以奶牛子宫为实验材料,刮取内膜上皮,用5%的冰乙酸抽提,通过酸性尿素聚丙烯酰胺电泳洗脱技术和反相高效液相色谱技术,分离纯化奶牛子宫黏液内源性抗菌肽,并进行其抗菌活性的检测。活性检测结果表明:在5min收集的样品没有抑菌圈出现,在45min收集的样品具有抗大肠杆菌BL21和金黄色葡萄球菌26003的活性。
奶牛子宫内膜防御素的发现有利于对奶牛黏膜的防御机制进行进一步的研究,从而为奶牛子宫内膜炎防治奠定了基础。
Endogenous antimicrobial peptides are an important component in innate immunity of organism defensins are a kind of positive Endogenous antimicrobial peptides, which contain cysteine-rich and distribute abroad in kingdom of animals and plants. Defensins are a large family among cationic endogenous antimicrobial peptide. As a kind of polypeptide,controlled by specific genic code and induced under specific external conditions.Endogenous antimicrobial peptides can resist the invasion of outside microorganism and kill inner mutant cells . According to the differences of position and connect manner of cysteines,the property of precursors,the position of expression, defensins are divided into five types:α-defensins,β-defensins,θ-defensins, insect defensins and plant defensins,β-defensins are mainly proved to be a antimicrobial barrier bacause they distribute in mucosal epithelial cells of mammals and aves.
In this study ,we have used cow as experiment. As a pair of primers were designed and synthesized according to the sequence of antibacterial peptide gene published in GenBank. Total RNA was purified from the bovine endometrial cells with the total RNA isolation kit. Then the DNA fragment of bovine antibacterial peptide was amplified by RT-PCR. After gel purification,the RT–PCR product was cloned into a pMD18-T Vector System. The vector mixtures were transformed into competent high DH5a. Some positive clones were selected on the LB agar-plate with X-Gal,Amp and IPTG. Length of the inserted DNA fragment was confirmed using BamHⅠand HindⅢdigestion and PCR, then was sequenced. The result showed that the obtained 138bp DNA fragment is identical to the BNBD5 sequence registered in the GenBank. The cDNA fragment code 45 amino acid,which contained six invariantly aminothiopropionic acid residues. Compared to bovine antibacterial peptide,it shared the highest identity of 99.3% with the BNBD5.We analysised ORF sequences of the Endometrial cows and other species BNBD5 antimicrobial peptidesβ-defensin-5 by DNAStar MegAlign of computer sofeware, It indicated that the relationship of cow to Capra hircus(DQ532360) is the highest (80.4%),to theβ-defensins of Mus musculus (NM_030734))and Rattus norvegicus (NM_001037549)is middle(56.5%,31.9%),to theβ-defensins of Anopheles gambiae (EU273600),Apis cerana cerana(EU727272) and Gallus gallus (AY621320, AY621307, DQ677636) is the lowest (mostly less than 20%), This shows thatβ-defensins of ruminants are species-specific.
And then target genes ,that was fragments of cow which coded the BNBD5 (138bp), were amplified by PCR with a pair of specifically expessive primers and recombinant plasmid as a template respectively, then cloned into pET28a expession vector. We Constructed prokaryotic expression plasmid pET-BNBD5 and transformed into Escherichia coli BL21.Established highly expression target protein inducing by IPTG. Protein molecular weight is about 10 KDa. in coli BL21 with the same size of expection . the best conditions of inducting were detemined that the induction time was six hours,the temperature was 37℃,and the IPTG Concentration was0.5mM. Under these conditions,the expressed recombinant protein existed in the form of inclusion bodies. By detecting the expressed recombinant protein with the method of agar geldiffusion test,theβ-defensins from the endometrial epithelial of cow , expressed in E. coli ,showed that antibacterial activity is not obvious in vitro.
The cow was involved in the experiment. Endometrial epithelial was isolated and acid soluble extract of cow was obtained and antibacterial activity was detected by gel overlay technique , Then it was further isolated and purified by HPLC and analyzed antibacterial activity by agarose radial diffusion assay.The molecular weight was determined by Tricine-SDS-PAGE. The result showed that it was able to effectively kill E. coliBL21 and the Staphylococcus bacteria 26003, its molecular weight was about 14KDa.
The discovery of BNBD5 of Endometrial of cow provides evidence for understanding the mucosal defense mechanism and laid the foundation of preventing and curing the endometritis of cow .
引文
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