猪XCL1基因原核表达、多克隆抗体制备及产物活性鉴定
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摘要
趋化因子(chemokines, chemoattractant cytokines)是能够使细胞定向移动的小分子细胞因子,在促使细胞迁移和活化过程中发挥重要作用,对机体的免疫系统非常重要。淋巴细胞趋化因子(Lymphotactin,Lptn)是C族趋化因子的一员,也被称为淋巴趋化因子配体1(XCL1)。淋巴趋化因子受体XCR1与XCL1相互作用参与呈递抗原、激活T淋巴细胞和NK细胞,发挥机体的细胞免疫功能,能够调节免疫系统平衡,增强粘膜免疫、抗肿瘤免疫,在感染性疾病过程中诱发炎症反应等。
柳超等已构建pEGFP-PLptn真核表达载体,证实绿色荧光融合蛋白在体外具有趋化活性,但并未进行进一步的研究。本研究首次构建了原核表达载体pET-32a-XCL1,摸索了原核表达的最佳诱导和纯化条件,采用HiTrap~(TM) Chelating HP纯化蛋白,SDS-PAGE检测重组蛋白纯化情况,Western-blot检测重组蛋白表达情况。并以此重组蛋白作为抗原,制备多克隆抗体,双向琼脂扩散试验、间接ELISA检测多克隆抗体效价,MTT法检测猪XCL1对淋巴细胞增殖的影响,为研究XCL1在猪中的功能提供了素材。主要研究内容与结果如下:
1.根据NCBI上猪XCL1基因序列,设计引物,从猪淋巴结克隆得到0.27 kb的目的片段。将目的片段克隆至pMD-19T载体中,筛选出阳性质粒送测序,结果表明该目的片段序列正确。
2.将目的基因构建到原核表达载体pET-32a (+),在大肠杆菌BL21(DE3)中进行原核表达,纯化目的蛋白,免疫新西兰大白兔,制备多克隆抗体。
3.双向琼脂扩散试验显示抗原抗体的结合比为1:8,间接ELISA检测到抗体效价达到1: 12 800。
4.通过MTT法试验检测重组蛋白XCL1对淋巴细胞增殖的影响,结果表明重组蛋白XCL1能促进淋巴细胞的增殖,而此实验制备的多克隆抗体可以阻断这种效应。
Chemokines(chemoattractant cytokines) are cytokines of small molecular size that can induce directed chemotaxis, and play an important role in the process of cell migration and activation. Thus, chemikines are very important components in the immune system. Lymphocyte chemotactic factor (Lymphotactin, Lptn) , also known as lymphoid chemokine ligand 1 (XCL1), is a member of C chemokine family. The interaction between XCR1 and XCL1 involves in antigen presentation, T lymphocytes and NK cells activation, and the cellular immune function exertion processes, thus can regulate immune balance and induce inflammatory reaction in the process of infectious disease, and can enhance mucosal immunity and anti-tumor immunity.
Liu et al(2010)had constructed eukaryotic expression vector pEGFP-PLptn, and confirmed that the fusion protein with green fluorescent marker displays chemotactic characteristic in vitro, but further study was not carried out. In this study, we first ever constructed prokaryotic expression vector pET-32a-XCL1 and found out the optimum conditions of its expression and purification. First, purify the recombinant protein using HiTrap~(TM) Chelating HP chromatographic column. Second, check the purified product using SDS-PAGE technique. Thirdly, detect the expression of the recombinant protein using western-blot.
Immunize the experimental animals (New Zealand white rabbits) with the purified fusion protein to prepare the serum that contains the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titre. Then, test the condition of lymphocytes proliferation by MTT. This research can provide basic material for further investigation of the function of XCL1 in swine. The main contents and results of our research are as follows:
1. Primers were designed according to XCL1 gene sequences on NCBI , and its ORF of about 0.27kb was amplified from the porcine lymph nodes and then integrated into the pMD-19T vector, and its positive plasmids were screened and sequenced. Sequencing results confirmed the correctness of the ORF in the recombinant vector.
2. The ORF of XCL1 gene was integrated into prokaryotic expression vector pET-32a (+), and expressed in E.coli.BL21(DE3). Immunize the New Zealand white rabbits with the purified fusion protein to prepare the serum that contains the associated polyclonal antibody.
3. Double immunodiffusion test showed the antigen-antibody binding ratio to be 1:8, and the titre of antibodies was 1: 12 800 detected by indirect ELISA.
4. MTT method was used to study how this recombinant protein affects on lymphocytes proliferation. This result of MTT showed that the recombinant protein could stimulate lymphocytes proliferation, and that this stimulating effect could be effectively blocked by the polyclonal antibody we prepared.
柳超等已构建pEGFP-PLptn真核表达载体,证实绿色荧光融合蛋白在体外具有趋化活性,但并未进行进一步的研究。本研究首次构建了原核表达载体pET-32a-XCL1,摸索了原核表达的最佳诱导和纯化条件,采用HiTrap~(TM) Chelating HP纯化蛋白,SDS-PAGE检测重组蛋白纯化情况,Western-blot检测重组蛋白表达情况。并以此重组蛋白作为抗原,制备多克隆抗体,双向琼脂扩散试验、间接ELISA检测多克隆抗体效价,MTT法检测猪XCL1对淋巴细胞增殖的影响,为研究XCL1在猪中的功能提供了素材。主要研究内容与结果如下:
1.根据NCBI上猪XCL1基因序列,设计引物,从猪淋巴结克隆得到0.27 kb的目的片段。将目的片段克隆至pMD-19T载体中,筛选出阳性质粒送测序,结果表明该目的片段序列正确。
2.将目的基因构建到原核表达载体pET-32a (+),在大肠杆菌BL21(DE3)中进行原核表达,纯化目的蛋白,免疫新西兰大白兔,制备多克隆抗体。
3.双向琼脂扩散试验显示抗原抗体的结合比为1:8,间接ELISA检测到抗体效价达到1: 12 800。
4.通过MTT法试验检测重组蛋白XCL1对淋巴细胞增殖的影响,结果表明重组蛋白XCL1能促进淋巴细胞的增殖,而此实验制备的多克隆抗体可以阻断这种效应。
Chemokines(chemoattractant cytokines) are cytokines of small molecular size that can induce directed chemotaxis, and play an important role in the process of cell migration and activation. Thus, chemikines are very important components in the immune system. Lymphocyte chemotactic factor (Lymphotactin, Lptn) , also known as lymphoid chemokine ligand 1 (XCL1), is a member of C chemokine family. The interaction between XCR1 and XCL1 involves in antigen presentation, T lymphocytes and NK cells activation, and the cellular immune function exertion processes, thus can regulate immune balance and induce inflammatory reaction in the process of infectious disease, and can enhance mucosal immunity and anti-tumor immunity.
Liu et al(2010)had constructed eukaryotic expression vector pEGFP-PLptn, and confirmed that the fusion protein with green fluorescent marker displays chemotactic characteristic in vitro, but further study was not carried out. In this study, we first ever constructed prokaryotic expression vector pET-32a-XCL1 and found out the optimum conditions of its expression and purification. First, purify the recombinant protein using HiTrap~(TM) Chelating HP chromatographic column. Second, check the purified product using SDS-PAGE technique. Thirdly, detect the expression of the recombinant protein using western-blot.
Immunize the experimental animals (New Zealand white rabbits) with the purified fusion protein to prepare the serum that contains the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titre. Then, test the condition of lymphocytes proliferation by MTT. This research can provide basic material for further investigation of the function of XCL1 in swine. The main contents and results of our research are as follows:
1. Primers were designed according to XCL1 gene sequences on NCBI , and its ORF of about 0.27kb was amplified from the porcine lymph nodes and then integrated into the pMD-19T vector, and its positive plasmids were screened and sequenced. Sequencing results confirmed the correctness of the ORF in the recombinant vector.
2. The ORF of XCL1 gene was integrated into prokaryotic expression vector pET-32a (+), and expressed in E.coli.BL21(DE3). Immunize the New Zealand white rabbits with the purified fusion protein to prepare the serum that contains the associated polyclonal antibody.
3. Double immunodiffusion test showed the antigen-antibody binding ratio to be 1:8, and the titre of antibodies was 1: 12 800 detected by indirect ELISA.
4. MTT method was used to study how this recombinant protein affects on lymphocytes proliferation. This result of MTT showed that the recombinant protein could stimulate lymphocytes proliferation, and that this stimulating effect could be effectively blocked by the polyclonal antibody we prepared.
引文
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