人N-乙酰半乳糖胺转移酶Ⅲ（huGALNT3）的异源表达及抗体制备
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摘要
糖链及其缀合物具有重要的生理作用和病理意义。肿瘤细胞中的糖链与正常细胞相比有明显变化。糖链的合成没有模板,而是通过一系列定位有序的糖基转移酶完成的,糖基转移酶表达差异将直接导致糖链结构的变化。因此,从糖基转移酶入手可以从本质上阐明糖链在肿瘤发生和发展过程中的变化规律及调控作用。N-乙酰半乳糖胺转移酶3(UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, ppGalNAc-T3, GALNT3)是GalNAc转移酶家族成员之一。该家族催化O-糖链形成过程中的第一步反应,即将GalNAc转移到多肽链的Ser/Thr残基上。家族成员之间在活性高低和底物选择性上有所不同。其中,GALNT3的活性与富含O-糖链的粘蛋白的形成关系密切,后者的异常是发生于多种上皮来源肿瘤的普遍现象。
本论文首先从人胃癌细胞MKN45扩增出GALNT3的cDNA,构建了大肠杆菌表达载体,并进行了表达。在大肠杆菌中使用可溶性和包涵体两种形式,成功实现了GALNT3可溶性区域(GALNT3-sol)的高效表达,并进行了纯化。经过活性测定发现,两种形式表达得到的GALNT3均无活性,原因可能是大肠杆菌缺少真核生物的蛋白合成后的修饰机制,而GALNT3是来源于人的一个复杂糖蛋白,异源表达产物未能形成有活性的天然构象。
另外,本实验还使用大肠杆菌表达的GALNT3-sol作为免疫原制备了抗GALNT3的抗体。将包涵体变性复性并纯化后SDS-PAGE分离,切胶免疫BALB/C小鼠,分别取第二次和第四次免疫后的血清,采用酶联免疫吸附检测方法(ELISA)检测抗体效价,二免的抗血清终点滴度为1：3200,而四免的抗血清终点滴度为1：25600。然后使用Western Blot方法鉴定所获抗体,阳性抗血清能够结合GALNT3-sol,使其显色,而阴性对照不能结合显色。以上结果显示,本实验所做的抗GALNT3-sol抗体具有较高的效价并能用于Western Blot检测,为进一步开发抗GALNT3的单克隆抗体,并进行临床材料的研究奠定了基础。
为了获得有活性的GALNT3蛋白,本论文又尝试了在巴斯德毕赤氏酵母(Pichia pastoris)中表达GALNT3。将GALNT3可溶性蛋白部分的基因(GALNT3-sol)克隆到pPIC9k质粒上,电转化进入Pichia pastoris GS115菌株。经过缺陷培养基,酵母菌体PCR和梯度抗生素(G418)等方法筛选获得高拷贝重组菌株。经过甲醇诱导GALNT3的表达,Western Blot结果证明GALNT3在毕赤酵母中成功表达,最佳表达条件为20℃,诱导72小时。对获得的GALNT3-sol进行活性检测,断定毕赤酵母表达的GALNT3-sol具有转糖基活性,这为进一步研究GALNT3对糖核苷酸或受体多肽的选择性奠定了基础。
总之,本论文在两种微生物体系中成功地表达了huGALNT3-sol,并首次使用大肠杆菌表达的GALNT3-sol制备了高效的抗体,同时首次在微生物中表达出了有活性的GALNT3,为在酵母真核表达体系中生产人类其他N-乙酰半乳糖胺转移酶,甚至其他类型糖基转移酶提出了可能。GALNTs活性蛋白的表达将为体外起始O-糖基化提供新的手段,为在药物蛋白上添加具有特殊生物学功能或活性的化合物提供框架。
Glycans and glycoconjugates play an important role in the occurrence and development of diseases. Great changes can be seen in glycans between Cancer cell and normal cells. There are no templates for the glycans synthesis, but through a series of orderly arranged glycosyltransferases completed. Differences in glycosyltransferase expression will directly result in changes in the structure of glycans. Therefore, the research about the glycosyltransferase could essentially clarify the glycan changes and the regulation of glycans in the process of tumor occurrence and development. UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (ppGalNAc-T3, GALNT3) has been proposed to be one memeber of the GalNAc transferase family. This family catalyses the transference of GalNAc to Ser/Thr residues in peptide, which is the first step in the formation of O-glycan. Different members of the family have various enzyme activity and substrate selectivity. And the activity of GALNT3 is closely related with the formation of mucin-type O-glycans, whose abnormity is found universally in epithelium-derived tumors.
Due to the initial study on the function of GalNAc transferases, firstly our research isolated cDNA of GALNT3 from MKN45 cell line, and then constructed expression vectors and expressed the protein in E. coli and Pichia Pastoris respectively.In E. coli the soluble region of GALNT3 without the hydrophobic transmembrane domain (GALNT3-sol) was expressed efficiently and purified as both soluble protein and inclusion body. The result of activity assay showed that GALNT3 obtained as both two forms had no activity, and the reason may lie in E. coli which lacks the protein modification mechanism of Eukaryotes (such as glycosylation, acetylation, phosphorylation, and disulfide bond formation). GALNT3 is a complex glycoprotein derived from Homo sapiens containing several disulfide bonds, so the GALNT3 expressed in E. coli cannot form natural and active conformation.
Besides, Our research also used GALNT3-sol inclusion body as immunogen to produce anti-GALNT3 antibody. After protein denaturation, refolding and purification, we isolated GALNT3-sol trough SDS-PAGE and cut off the gel containing the protein to inject BALB/C mice. Then the serum after the second and forth immunization was collected respectively, and the antibody titer was tested by ELISA. The antibody titer after the second immunization was 1:3200, and the titer after the forth was 1:25600. Subsequently the antibody was tested by Western Blot, and the positive serum could combine and show the protein GALNT3-sol, while the negative serum couldn't. These results reveal that the anti-GALNT3-sol antibody has high titer and could be used in Western Blot, which may prove important in the development of commercial anti-GALNT3 monoclonal antibody used in clinical assay.
To obtain active GALNT3, we tried to express it in Pichia pastoris. The gene of GALNT3-sol was cloned into the plasmid pPIC9K, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains were screened out after defect medium, yeast PCR and gradient antibiotic (G418) and so on. The expression of GALNT3-sol was confirmed through Western Blot after methanol induction. The expression of GALNT3-sol was best induced for 72 hours at 20℃in Pichia pastoris. After the concentration of culture medium, the enzyme activity was tested. The results show that GALNT3-sol expressed in Pichia pastoris has the activity of transferring GalNAc to Ser/Thr residues in peptide, which provide support for the further research on the substrate specificity (sugar nucleotide or acceptor peptide) of GALNT3.
Our research expressed massive GALNT3-sol protein in microbes successfully. The efficient polyclonal antibody was prepared using GALNT3-sol expressed in E. coli as immunogen for the first time, and furthermore it is also the first active GALNT3 protein expressed in microbes which make the production of other human ppGalNAc-Ts and ever other various glycotransferases in Pichia pastoris possible. The expression of active GALNTs will provide new means for initiating O-glycosylation and new platform for attaching compounds with special biological function or activity to therapeutic proteins.
本论文首先从人胃癌细胞MKN45扩增出GALNT3的cDNA,构建了大肠杆菌表达载体,并进行了表达。在大肠杆菌中使用可溶性和包涵体两种形式,成功实现了GALNT3可溶性区域(GALNT3-sol)的高效表达,并进行了纯化。经过活性测定发现,两种形式表达得到的GALNT3均无活性,原因可能是大肠杆菌缺少真核生物的蛋白合成后的修饰机制,而GALNT3是来源于人的一个复杂糖蛋白,异源表达产物未能形成有活性的天然构象。
另外,本实验还使用大肠杆菌表达的GALNT3-sol作为免疫原制备了抗GALNT3的抗体。将包涵体变性复性并纯化后SDS-PAGE分离,切胶免疫BALB/C小鼠,分别取第二次和第四次免疫后的血清,采用酶联免疫吸附检测方法(ELISA)检测抗体效价,二免的抗血清终点滴度为1：3200,而四免的抗血清终点滴度为1：25600。然后使用Western Blot方法鉴定所获抗体,阳性抗血清能够结合GALNT3-sol,使其显色,而阴性对照不能结合显色。以上结果显示,本实验所做的抗GALNT3-sol抗体具有较高的效价并能用于Western Blot检测,为进一步开发抗GALNT3的单克隆抗体,并进行临床材料的研究奠定了基础。
为了获得有活性的GALNT3蛋白,本论文又尝试了在巴斯德毕赤氏酵母(Pichia pastoris)中表达GALNT3。将GALNT3可溶性蛋白部分的基因(GALNT3-sol)克隆到pPIC9k质粒上,电转化进入Pichia pastoris GS115菌株。经过缺陷培养基,酵母菌体PCR和梯度抗生素(G418)等方法筛选获得高拷贝重组菌株。经过甲醇诱导GALNT3的表达,Western Blot结果证明GALNT3在毕赤酵母中成功表达,最佳表达条件为20℃,诱导72小时。对获得的GALNT3-sol进行活性检测,断定毕赤酵母表达的GALNT3-sol具有转糖基活性,这为进一步研究GALNT3对糖核苷酸或受体多肽的选择性奠定了基础。
总之,本论文在两种微生物体系中成功地表达了huGALNT3-sol,并首次使用大肠杆菌表达的GALNT3-sol制备了高效的抗体,同时首次在微生物中表达出了有活性的GALNT3,为在酵母真核表达体系中生产人类其他N-乙酰半乳糖胺转移酶,甚至其他类型糖基转移酶提出了可能。GALNTs活性蛋白的表达将为体外起始O-糖基化提供新的手段,为在药物蛋白上添加具有特殊生物学功能或活性的化合物提供框架。
Glycans and glycoconjugates play an important role in the occurrence and development of diseases. Great changes can be seen in glycans between Cancer cell and normal cells. There are no templates for the glycans synthesis, but through a series of orderly arranged glycosyltransferases completed. Differences in glycosyltransferase expression will directly result in changes in the structure of glycans. Therefore, the research about the glycosyltransferase could essentially clarify the glycan changes and the regulation of glycans in the process of tumor occurrence and development. UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (ppGalNAc-T3, GALNT3) has been proposed to be one memeber of the GalNAc transferase family. This family catalyses the transference of GalNAc to Ser/Thr residues in peptide, which is the first step in the formation of O-glycan. Different members of the family have various enzyme activity and substrate selectivity. And the activity of GALNT3 is closely related with the formation of mucin-type O-glycans, whose abnormity is found universally in epithelium-derived tumors.
Due to the initial study on the function of GalNAc transferases, firstly our research isolated cDNA of GALNT3 from MKN45 cell line, and then constructed expression vectors and expressed the protein in E. coli and Pichia Pastoris respectively.In E. coli the soluble region of GALNT3 without the hydrophobic transmembrane domain (GALNT3-sol) was expressed efficiently and purified as both soluble protein and inclusion body. The result of activity assay showed that GALNT3 obtained as both two forms had no activity, and the reason may lie in E. coli which lacks the protein modification mechanism of Eukaryotes (such as glycosylation, acetylation, phosphorylation, and disulfide bond formation). GALNT3 is a complex glycoprotein derived from Homo sapiens containing several disulfide bonds, so the GALNT3 expressed in E. coli cannot form natural and active conformation.
Besides, Our research also used GALNT3-sol inclusion body as immunogen to produce anti-GALNT3 antibody. After protein denaturation, refolding and purification, we isolated GALNT3-sol trough SDS-PAGE and cut off the gel containing the protein to inject BALB/C mice. Then the serum after the second and forth immunization was collected respectively, and the antibody titer was tested by ELISA. The antibody titer after the second immunization was 1:3200, and the titer after the forth was 1:25600. Subsequently the antibody was tested by Western Blot, and the positive serum could combine and show the protein GALNT3-sol, while the negative serum couldn't. These results reveal that the anti-GALNT3-sol antibody has high titer and could be used in Western Blot, which may prove important in the development of commercial anti-GALNT3 monoclonal antibody used in clinical assay.
To obtain active GALNT3, we tried to express it in Pichia pastoris. The gene of GALNT3-sol was cloned into the plasmid pPIC9K, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains were screened out after defect medium, yeast PCR and gradient antibiotic (G418) and so on. The expression of GALNT3-sol was confirmed through Western Blot after methanol induction. The expression of GALNT3-sol was best induced for 72 hours at 20℃in Pichia pastoris. After the concentration of culture medium, the enzyme activity was tested. The results show that GALNT3-sol expressed in Pichia pastoris has the activity of transferring GalNAc to Ser/Thr residues in peptide, which provide support for the further research on the substrate specificity (sugar nucleotide or acceptor peptide) of GALNT3.
Our research expressed massive GALNT3-sol protein in microbes successfully. The efficient polyclonal antibody was prepared using GALNT3-sol expressed in E. coli as immunogen for the first time, and furthermore it is also the first active GALNT3 protein expressed in microbes which make the production of other human ppGalNAc-Ts and ever other various glycotransferases in Pichia pastoris possible. The expression of active GALNTs will provide new means for initiating O-glycosylation and new platform for attaching compounds with special biological function or activity to therapeutic proteins.
引文
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