绵羊CCL28和Granulysin基因的克隆与体外表达
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摘要
CC趋化因子配体28(CCL28),又称黏膜相关上皮趋化因子(MEC),是新近发现的CC趋化因子家族成员,在人和小鼠的大部分黏膜组织(唾液腺、乳腺、小肠、大肠和气管)中表达,并且是由以上组织的上皮细胞产生。CCL28对IgA抗体分泌细胞(IgA-ASCs)的归巢起重要作用,此外,CCL28还具有广谱的抗微生物活性。
Granulysin是一种表达于人体细胞毒性T淋巴细胞(CTL)和自然杀伤细胞(NK Cells)中的一种抗菌蛋白,属于SAPLIP(saposin-like protein)家族的一类脂封闭蛋白,是目前CTL胞浆颗粒中唯一证实具有抗菌活性的效应分子。Granulysin能与穿孔素和颗粒酶共同定位于人的细胞毒性T淋巴细胞和自然杀伤细胞颗粒中,其作用相当于广谱抗菌素,作用范围包括革兰氏阳性菌、革兰氏阴性菌、真菌和寄生虫,Granulysin也能杀灭人的肿瘤细胞。
为了研究CCL28和Granulysin在绵羊体内是否具有同样的功能,我们克隆了绵羊CCL28和Granulysin基因,并得到了相应的融合蛋白,试验分两个系列。
一、绵羊CCL28基因的克隆与体外表达。利用同源序列克隆原理设计一对克隆引物,对绵羊结肠黏膜组织提取的总RNA进行RT-PCR,将PCR产物与pMD19-T载体连接后转化Ecol.JM109感受态细胞,筛选阳性克隆、测序,并进行序列分析;又根据克隆序列设计一对表达引物,用PCR法从重组克隆载体中扩增出含BamHI/XhoI酶切位点的CCL28片段,双酶切后亚克隆至pGEX-4T-1载体,构建重组原核表达载体pGEX-CCL28,转化宿主菌E.coli BL21,经IPTG诱导进行表达,SDS-PAGE鉴定。克隆的绵羊CCL28基因包含完整的开放阅读框架,长为444bp,ORF为387bp,编码129个氨基酸,与人、小鼠、猪和牛该基因的同源性分别为76.4%、61.4%、84.3%和92.9%,推导的氨基酸序列与人、小鼠、猪和牛的同源性分别为76%、63%、84%和93%,信号肽为1~24aa,SCY功能域为27~88aa,结构特征与人、小鼠、猪和牛的CCL28相一致,成功构建了原核表达载体pGEX-CCL28,表达的融合蛋白分子量约为39ku,为进一步研究绵羊CCL28的生物学功能奠定了基础。
二、绵羊Granulysin基因的克隆与体外表达。基于电子延伸序列,设计一对克隆引物,对绵羊回肠黏膜组织提取的总RNA进行RT-PCR,将PCR产物与pMD19-T载体连接后转化Ecol.JM109感受态细胞,筛选阳性克隆、测序,并进行序列分析;又根据克隆序列设计一对表达引物,用PCR法从重组克隆质粒中扩增出含BamHI/XhoI酶切位点的Granulysin片段,双酶切后亚克隆至pGEX-4T-1载体,构建重组原核表达载体pGEX-Granulysin,转化宿主菌E.coli BL21,经IPTG诱导进行表达,SDS-PAGE鉴定。克隆的绵羊Granulysin基因包含完整的开放阅读框架,长为509bp,ORF为438bp,编码146个氨基酸,与人和牛该基因的同源性分别为56.2%和87.2%,推导的氨基酸序列与人和牛的同源性分别为38%和75%,信号肽为1~22aa,SapB功能域为64~138aa,结构特征与人和牛的Granulysin相一致,成功构建了原核表达载体pGEX-CCL28,表达的融合蛋白分子量约为41 ku,为进一步研究绵羊Granulysin的生物学功能奠定了基础。
CC chemokine ligand 28 (CCL28), also called mucosae-associated epithelial chemokine (MEC), is the member of the CC chemokine subfamily which was discovered recently and expressed in most mucosal tissues of human and mouse which include the salivary gland, mammary gland, small and large intestines and trachea, where it is predominantly produced by epithelial cells. CCL28 play a essential role in homing of IgA antibody secreting cell (IgA-ASCs), and CCL28 also has a broad-spectrum antimicrobial activity.
Granulysin, a antimicrobial protein expressed in Cytolytic T Lymphocytes (CTL) and Natural Killer cells (NK cells), is a member of the saposin-like protein (SAPLIP) family of lipid binding proteins and the only one antimicrobial effector molecule which was identified in CTL endochylema at present. Granulysin can colocalizes in human CTL and NK cells with perforin and granzymes. Its function is similar to broad-spectrum antibiotics against both Gram-positive and Gram-negative bacteria, fungi, and parasites. Granulysin also kills human tumor cells.
To investigate if CCL28 and Granulysin play same roles in the sheep, we cloned ovine CCL28 and Granulysin and obtained cognate fusion protein. The study was divided into two series.
Ⅰ. Cloning and expression of ovine CCL28 in vitro. A pair of cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from mucosal tissue of ovine colon was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of ovine CCL28 cDNA containing BamHI/XhoI was amplified from recombinant vector by PCR. The fragment digested by BamHI/XhoI was subcloned into pGEX-4T-1 vector to construct a recombinant prokaryotic expression vector, pGEX-CCL28. Subsequently, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The cloned sequences containing the open reading frame of ovine CCL28 consist of 444bp, and ORF is 387bp and encodes 129 amino acids. Identity analysis showed that the ovine CCL28 nucleotide sequences shared 76.4%, 61.4%, 84.3% and 92.9% homology with that of human, mouse, pig and cattle, the deduced amino acid sequences shared 76%, 63%, 84% and 93% homology with that of human, mouse, pig and cattle. The signal peptide is 1-24aa and the domain SCY is 27-88aa, which reveals that the structural feature is consistent with human, mouse pig and cattle. The prokaryotic expression pGEX-CCL28 was successfully constructed. The fusion protein expressed in E.coli BL21 was about 39ku. It provides the experiment basis for further researching its biological function.
Ⅱ. Cloning and expression of ovine Granulysin in vitro. A pair of cloning primers were designed based on the in silico sequence information. Total RNA extracted from mucosal tissue of ovine ileum was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of ovine Granulysin cDNA containing BamHI/XhoI was amplified from recombinant vector by PCR. The fragment digested by BamHI/XhoI was subcloned into pGEX-4T-1 vector to construct a recombinant prokaryotic expression vector, pGEX- Granulysin. Subsequently, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The cloned sequence containing the open reading frame of ovine Granulysin consists of 509bp, and ORF is 438bp and encodes 146 amino acids. Identity analysis showed that the ovine Granulysin nucleotide sequence shared 56.2% and 87.2% homology with that of human and cattle, the predicted amino acid shared 38% and 75% homology with that of human and cattle. The signal peptide is 1-22aa and the domain SapB is 64-138aa, which reveals that the structural feature is consistent with human and cattle. The prokaryotic expression pGEX-Granulysin was successfully constructed. The fusion protein expressed in E.coli BL21 was about 41ku. It provides the experiment basis for further researching its biological function.
Granulysin是一种表达于人体细胞毒性T淋巴细胞(CTL)和自然杀伤细胞(NK Cells)中的一种抗菌蛋白,属于SAPLIP(saposin-like protein)家族的一类脂封闭蛋白,是目前CTL胞浆颗粒中唯一证实具有抗菌活性的效应分子。Granulysin能与穿孔素和颗粒酶共同定位于人的细胞毒性T淋巴细胞和自然杀伤细胞颗粒中,其作用相当于广谱抗菌素,作用范围包括革兰氏阳性菌、革兰氏阴性菌、真菌和寄生虫,Granulysin也能杀灭人的肿瘤细胞。
为了研究CCL28和Granulysin在绵羊体内是否具有同样的功能,我们克隆了绵羊CCL28和Granulysin基因,并得到了相应的融合蛋白,试验分两个系列。
一、绵羊CCL28基因的克隆与体外表达。利用同源序列克隆原理设计一对克隆引物,对绵羊结肠黏膜组织提取的总RNA进行RT-PCR,将PCR产物与pMD19-T载体连接后转化Ecol.JM109感受态细胞,筛选阳性克隆、测序,并进行序列分析;又根据克隆序列设计一对表达引物,用PCR法从重组克隆载体中扩增出含BamHI/XhoI酶切位点的CCL28片段,双酶切后亚克隆至pGEX-4T-1载体,构建重组原核表达载体pGEX-CCL28,转化宿主菌E.coli BL21,经IPTG诱导进行表达,SDS-PAGE鉴定。克隆的绵羊CCL28基因包含完整的开放阅读框架,长为444bp,ORF为387bp,编码129个氨基酸,与人、小鼠、猪和牛该基因的同源性分别为76.4%、61.4%、84.3%和92.9%,推导的氨基酸序列与人、小鼠、猪和牛的同源性分别为76%、63%、84%和93%,信号肽为1~24aa,SCY功能域为27~88aa,结构特征与人、小鼠、猪和牛的CCL28相一致,成功构建了原核表达载体pGEX-CCL28,表达的融合蛋白分子量约为39ku,为进一步研究绵羊CCL28的生物学功能奠定了基础。
二、绵羊Granulysin基因的克隆与体外表达。基于电子延伸序列,设计一对克隆引物,对绵羊回肠黏膜组织提取的总RNA进行RT-PCR,将PCR产物与pMD19-T载体连接后转化Ecol.JM109感受态细胞,筛选阳性克隆、测序,并进行序列分析;又根据克隆序列设计一对表达引物,用PCR法从重组克隆质粒中扩增出含BamHI/XhoI酶切位点的Granulysin片段,双酶切后亚克隆至pGEX-4T-1载体,构建重组原核表达载体pGEX-Granulysin,转化宿主菌E.coli BL21,经IPTG诱导进行表达,SDS-PAGE鉴定。克隆的绵羊Granulysin基因包含完整的开放阅读框架,长为509bp,ORF为438bp,编码146个氨基酸,与人和牛该基因的同源性分别为56.2%和87.2%,推导的氨基酸序列与人和牛的同源性分别为38%和75%,信号肽为1~22aa,SapB功能域为64~138aa,结构特征与人和牛的Granulysin相一致,成功构建了原核表达载体pGEX-CCL28,表达的融合蛋白分子量约为41 ku,为进一步研究绵羊Granulysin的生物学功能奠定了基础。
CC chemokine ligand 28 (CCL28), also called mucosae-associated epithelial chemokine (MEC), is the member of the CC chemokine subfamily which was discovered recently and expressed in most mucosal tissues of human and mouse which include the salivary gland, mammary gland, small and large intestines and trachea, where it is predominantly produced by epithelial cells. CCL28 play a essential role in homing of IgA antibody secreting cell (IgA-ASCs), and CCL28 also has a broad-spectrum antimicrobial activity.
Granulysin, a antimicrobial protein expressed in Cytolytic T Lymphocytes (CTL) and Natural Killer cells (NK cells), is a member of the saposin-like protein (SAPLIP) family of lipid binding proteins and the only one antimicrobial effector molecule which was identified in CTL endochylema at present. Granulysin can colocalizes in human CTL and NK cells with perforin and granzymes. Its function is similar to broad-spectrum antibiotics against both Gram-positive and Gram-negative bacteria, fungi, and parasites. Granulysin also kills human tumor cells.
To investigate if CCL28 and Granulysin play same roles in the sheep, we cloned ovine CCL28 and Granulysin and obtained cognate fusion protein. The study was divided into two series.
Ⅰ. Cloning and expression of ovine CCL28 in vitro. A pair of cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from mucosal tissue of ovine colon was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of ovine CCL28 cDNA containing BamHI/XhoI was amplified from recombinant vector by PCR. The fragment digested by BamHI/XhoI was subcloned into pGEX-4T-1 vector to construct a recombinant prokaryotic expression vector, pGEX-CCL28. Subsequently, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The cloned sequences containing the open reading frame of ovine CCL28 consist of 444bp, and ORF is 387bp and encodes 129 amino acids. Identity analysis showed that the ovine CCL28 nucleotide sequences shared 76.4%, 61.4%, 84.3% and 92.9% homology with that of human, mouse, pig and cattle, the deduced amino acid sequences shared 76%, 63%, 84% and 93% homology with that of human, mouse, pig and cattle. The signal peptide is 1-24aa and the domain SCY is 27-88aa, which reveals that the structural feature is consistent with human, mouse pig and cattle. The prokaryotic expression pGEX-CCL28 was successfully constructed. The fusion protein expressed in E.coli BL21 was about 39ku. It provides the experiment basis for further researching its biological function.
Ⅱ. Cloning and expression of ovine Granulysin in vitro. A pair of cloning primers were designed based on the in silico sequence information. Total RNA extracted from mucosal tissue of ovine ileum was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of ovine Granulysin cDNA containing BamHI/XhoI was amplified from recombinant vector by PCR. The fragment digested by BamHI/XhoI was subcloned into pGEX-4T-1 vector to construct a recombinant prokaryotic expression vector, pGEX- Granulysin. Subsequently, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The cloned sequence containing the open reading frame of ovine Granulysin consists of 509bp, and ORF is 438bp and encodes 146 amino acids. Identity analysis showed that the ovine Granulysin nucleotide sequence shared 56.2% and 87.2% homology with that of human and cattle, the predicted amino acid shared 38% and 75% homology with that of human and cattle. The signal peptide is 1-22aa and the domain SapB is 64-138aa, which reveals that the structural feature is consistent with human and cattle. The prokaryotic expression pGEX-Granulysin was successfully constructed. The fusion protein expressed in E.coli BL21 was about 41ku. It provides the experiment basis for further researching its biological function.
引文
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