鸡MHCⅡα/β链基因的克隆和原核表达及其抗体制备
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摘要
主要组织相容性复合体(Major histocompatibility complex,MHC)是由紧密连锁的高度多态的基因位点所组成的位于染色体上一个遗传区域,广泛存在于所有脊椎动物,具有高度的多态性和保守性。该基因区编码的蛋白通常称为MHC分子或MHC抗原,是移植物排斥的主要决定簇。根据结构和功能可将MHC分为Ⅰ、Ⅱ、Ⅲ三类。其中,MHC Ⅱ类分子是由2条非共价结合的多肽链(α链和β链)构成的糖蛋白,它们由不同的MHC基因编码。α链(32 000～34 000)比β链(29 000～32 000)略大,2条链均含有寡糖,α链糖基化程度较高。结构上Ⅰ类分子和Ⅱ类分子均可分成肽结合区、Ig样区、跨膜区和胞浆区4个部位。
MHC Ⅱ类分子选择性地分布于有免疫功能的细胞如B细胞、单核细胞、巨噬细胞、树突状细胞、激活的T细胞等细胞膜上。MHC的主要功能是递呈抗原肽段,通过MHC-抗原肽复合物活化、诱导T细胞和B细胞的免疫应答。MHC Ⅱ类分子递呈的抗原主要是外源性抗原。为了探索鸡MHCⅡ类分子在免疫应答中的作用及其机理,本研究运用分子生物学技术,进行了克隆、表达和鉴定。
首先,根据GenBank中登录的鸡类MHC Ⅱ的α链和β链基因序列与载体pGEX-47-1的多克隆酶切位点分别设计两对引物,利用反转录-聚合酶链式反应(R7-PCR)从有丝分裂原刀豆蛋白A(CortA)刺激18h活化的鸡脾淋巴细胞中扩增出相应大小的α链(771bp)和β链(798bp)的基因;经酶切鉴定,所扩增的α链和β链基因片段含有相应的酶切位点;然后将这两个片段分别插入载体pGEX-4T-1中,并转化宿主菌BL21,经双酶切鉴定重组质粒pGEX-4T-1/α和pGEX-4T-1/β后,对α链和β链的核苷酸序列进行测定。结果表明本研究成功地克隆了鸡MHCⅡ的α链和β链基因,它们分别编码771和798个核苷酸。与GeneBank登录的α链基因(登录号:AY357254)相比较,只有四个核苷酸的差异,同源性为99%,导致2个氨基酸的改变。同样,与GeneBank登录的β链基因(登录号:S66480)相比较,有42个核苷酸的差异,同源性为95%,导致27个氨基酸的改变。
其次,将上述构建成功的原核表达重组质粒pGEX-4T-1/α和pGEX-4T-1/β,转化大肠杆菌BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)于37℃诱导培养4h,表达出含有谷胱甘肽-S-转移酶(GST)的融合蛋白GST-α和GST-β;对表达条件进行相应的优化,确定了最佳诱导表达时间和诱导剂浓度;对表达蛋白的可溶性进行鉴定,结果表明表达的融合蛋白GST-β和GST-β均以包涵体形式存在;利用优化的原核表达条件对含有质粒GST-β和GST-β的BL21菌进行大量诱导表达,通过反复冻融和超声裂解诱导菌,获得浓度较高的GST-α和GST-β包涵体蛋白,用十二烷基肌氨酸钠加超声波的方法使包涵体充分溶解,溶解的包涵体通过谷胱甘肽琼脂糖亲和层析获得
The major histocompatibility complex (MHC) is an extended cluster of genes with extraordinary polymorphism and compact linkage in the chromosome, spreading over all vertebrates. MHC molecules are also considered as the primary determinants in the transplant rejection. According to their structure and function, MHC molecules could be divided into class Ⅰ, Ⅱ and Ⅲ. Class Ⅱ molecules are glycoproteins that are formed by two noncovalence-associated chains, a (32000~34000) and p (29000~32000), each of which is encoded by different MHC genes. Like class I molecules, class II molecules consist of peptide-binding region, immunoglobulin-like region, transmenbrane region and cytoplasmic region. Class Ⅱ molecules mainly distribute on the membrane of the maturing B cells, presenting cells (macrophage cells and dendritic cells) and activated T cells. These molecules have an effect on presenting antigen and activating T cells by forming class II molecule-antigen peptide complex, which can stimulate T cells and B cells to participate in immune response. However class Ⅱ molecules mostly present exogenous antigen. In order to research the function and mechanism of class MHC Ⅱ of chicken in immune response, we cloned, expressed and identified the gene of chain α and β of class Ⅱ.Firstly, according to mRNA gene sequence registered in GenBank and multi-cloning restriction enzyme sites of vector pGEX-4T-1, two pairs of specific primers for the genes of chain α and β of class II of chicken were designed and synthesized respectively. Using total RNA from ConA-stimulated chicken spleen lymphocytes, two genes about 771bp and 798bp were amplified by RT-PCR respectively. The results of endonuclease EcoRI digesting RT-PCR product of chain a and endonuclease PstI digesting RT-PCR product of chain β show that two genes have the corresponding restriction enzyme site as the known α and β gene. Then the genes were inserted into vector pGEX-4T-1 respectively, and the recombinant plasmids were transformed into E.coli BL21. The recombinant plasmid pGEX-4T-1/α was identified by BamHI+SalI digestion and the recombinant plasmid pGEX-4T-l/p was identified by EcoRI+SalI digestion. Then, the positive recombinant clone was sequenced and analyzed. The results show that the complete ORF of a gene contains 771 nucleic acids and encodes 256 amino acids; the nucleotide sequence similarity is approximately 99% with the reported a gene. Meanwhile, the complete ORF of β gene contains 798 nucleic acids and encodes 266 amino acids; the nucleotide sequence similarity is approximately 95% with the reported chicken β gene. These suggest that the a
and β genes of chicken MHC class II molecules have been successfully cloned from chicken spleen lymphocytes.Secondly, the constructed recombinant plasmids pGEX-4T-1/α and pGEX-4T-l/p were transformed into E.coli BL21 and then induced to express GST-a and GST-P fusion protein by IPTG at different concentration and at different times. After optimizing prokaryotic expression conditions, 4 h was determined as the optimum induction time and 1 mmol/L was determined as the concentration of IPTG. GST-a and GST-P fusion protein solubilities were identificated and the result indicated that expressed GST-a and GST-P protein were located in inclusion bodies. Under the optimal condition, the recombinant plasmids were induced to express large-scale GST-a and GST-P fusion proteins and the induced recombinant bacteria were lysed by freeze-thaw and sonication. The obtained GST-a and GST-P inclusion body proteins were solubilized by sonication with the adding of the detergent lauroylsarcosine. The solubilized GST-a and GST-p protein were purified by affinity chromatography with glutathione agarose.Finally, mice were immunizated with the purified GST-a and GST-P fusion proteins and the polyclonal antiserum against chain a and p of chicken were collected. A specific reaction appeared between the antibodies and GST-a and GST-P fusion protein in agar diffusion assay and in ELISA, these results indicated that fusion proteins, GST-a
MHC Ⅱ类分子选择性地分布于有免疫功能的细胞如B细胞、单核细胞、巨噬细胞、树突状细胞、激活的T细胞等细胞膜上。MHC的主要功能是递呈抗原肽段,通过MHC-抗原肽复合物活化、诱导T细胞和B细胞的免疫应答。MHC Ⅱ类分子递呈的抗原主要是外源性抗原。为了探索鸡MHCⅡ类分子在免疫应答中的作用及其机理,本研究运用分子生物学技术,进行了克隆、表达和鉴定。
首先,根据GenBank中登录的鸡类MHC Ⅱ的α链和β链基因序列与载体pGEX-47-1的多克隆酶切位点分别设计两对引物,利用反转录-聚合酶链式反应(R7-PCR)从有丝分裂原刀豆蛋白A(CortA)刺激18h活化的鸡脾淋巴细胞中扩增出相应大小的α链(771bp)和β链(798bp)的基因;经酶切鉴定,所扩增的α链和β链基因片段含有相应的酶切位点;然后将这两个片段分别插入载体pGEX-4T-1中,并转化宿主菌BL21,经双酶切鉴定重组质粒pGEX-4T-1/α和pGEX-4T-1/β后,对α链和β链的核苷酸序列进行测定。结果表明本研究成功地克隆了鸡MHCⅡ的α链和β链基因,它们分别编码771和798个核苷酸。与GeneBank登录的α链基因(登录号:AY357254)相比较,只有四个核苷酸的差异,同源性为99%,导致2个氨基酸的改变。同样,与GeneBank登录的β链基因(登录号:S66480)相比较,有42个核苷酸的差异,同源性为95%,导致27个氨基酸的改变。
其次,将上述构建成功的原核表达重组质粒pGEX-4T-1/α和pGEX-4T-1/β,转化大肠杆菌BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)于37℃诱导培养4h,表达出含有谷胱甘肽-S-转移酶(GST)的融合蛋白GST-α和GST-β;对表达条件进行相应的优化,确定了最佳诱导表达时间和诱导剂浓度;对表达蛋白的可溶性进行鉴定,结果表明表达的融合蛋白GST-β和GST-β均以包涵体形式存在;利用优化的原核表达条件对含有质粒GST-β和GST-β的BL21菌进行大量诱导表达,通过反复冻融和超声裂解诱导菌,获得浓度较高的GST-α和GST-β包涵体蛋白,用十二烷基肌氨酸钠加超声波的方法使包涵体充分溶解,溶解的包涵体通过谷胱甘肽琼脂糖亲和层析获得
The major histocompatibility complex (MHC) is an extended cluster of genes with extraordinary polymorphism and compact linkage in the chromosome, spreading over all vertebrates. MHC molecules are also considered as the primary determinants in the transplant rejection. According to their structure and function, MHC molecules could be divided into class Ⅰ, Ⅱ and Ⅲ. Class Ⅱ molecules are glycoproteins that are formed by two noncovalence-associated chains, a (32000~34000) and p (29000~32000), each of which is encoded by different MHC genes. Like class I molecules, class II molecules consist of peptide-binding region, immunoglobulin-like region, transmenbrane region and cytoplasmic region. Class Ⅱ molecules mainly distribute on the membrane of the maturing B cells, presenting cells (macrophage cells and dendritic cells) and activated T cells. These molecules have an effect on presenting antigen and activating T cells by forming class II molecule-antigen peptide complex, which can stimulate T cells and B cells to participate in immune response. However class Ⅱ molecules mostly present exogenous antigen. In order to research the function and mechanism of class MHC Ⅱ of chicken in immune response, we cloned, expressed and identified the gene of chain α and β of class Ⅱ.Firstly, according to mRNA gene sequence registered in GenBank and multi-cloning restriction enzyme sites of vector pGEX-4T-1, two pairs of specific primers for the genes of chain α and β of class II of chicken were designed and synthesized respectively. Using total RNA from ConA-stimulated chicken spleen lymphocytes, two genes about 771bp and 798bp were amplified by RT-PCR respectively. The results of endonuclease EcoRI digesting RT-PCR product of chain a and endonuclease PstI digesting RT-PCR product of chain β show that two genes have the corresponding restriction enzyme site as the known α and β gene. Then the genes were inserted into vector pGEX-4T-1 respectively, and the recombinant plasmids were transformed into E.coli BL21. The recombinant plasmid pGEX-4T-1/α was identified by BamHI+SalI digestion and the recombinant plasmid pGEX-4T-l/p was identified by EcoRI+SalI digestion. Then, the positive recombinant clone was sequenced and analyzed. The results show that the complete ORF of a gene contains 771 nucleic acids and encodes 256 amino acids; the nucleotide sequence similarity is approximately 99% with the reported a gene. Meanwhile, the complete ORF of β gene contains 798 nucleic acids and encodes 266 amino acids; the nucleotide sequence similarity is approximately 95% with the reported chicken β gene. These suggest that the a
and β genes of chicken MHC class II molecules have been successfully cloned from chicken spleen lymphocytes.Secondly, the constructed recombinant plasmids pGEX-4T-1/α and pGEX-4T-l/p were transformed into E.coli BL21 and then induced to express GST-a and GST-P fusion protein by IPTG at different concentration and at different times. After optimizing prokaryotic expression conditions, 4 h was determined as the optimum induction time and 1 mmol/L was determined as the concentration of IPTG. GST-a and GST-P fusion protein solubilities were identificated and the result indicated that expressed GST-a and GST-P protein were located in inclusion bodies. Under the optimal condition, the recombinant plasmids were induced to express large-scale GST-a and GST-P fusion proteins and the induced recombinant bacteria were lysed by freeze-thaw and sonication. The obtained GST-a and GST-P inclusion body proteins were solubilized by sonication with the adding of the detergent lauroylsarcosine. The solubilized GST-a and GST-p protein were purified by affinity chromatography with glutathione agarose.Finally, mice were immunizated with the purified GST-a and GST-P fusion proteins and the polyclonal antiserum against chain a and p of chicken were collected. A specific reaction appeared between the antibodies and GST-a and GST-P fusion protein in agar diffusion assay and in ELISA, these results indicated that fusion proteins, GST-a
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