中国近交系版纳微型猪α1,3-半乳糖基转移酶基因剪接变异体的克隆及其在人类细胞中的表达

目的克隆中国近交系版纳微型猪(Chinese Banna Minipig Inbred Line,BMI)的α1,3-半乳糖基转移酶(α1,3-GT)基因并分离其剪接变异体,构建其真核表达载体并观察BMIα1,3-GT基因在人肺腺癌A549细胞中的表达和功能。方法提取BMI肝组织总RNA,RT-PCR扩增全长的α1,3-GTcDNA,并克隆到pMD18-T载体,挑选15个阳性克隆进行测序,获得GT1、GT2两种基因剪接变异体。将GT1、GT2克隆到pEGFP-N1上构建其真核表达载体,分别命名为pN-GT1、pN-GT2。将pN-GT1、pN-GT2分别转染人肺腺癌A549细胞,RT-PCR检测转染细胞中α1,3-GT mRNA的表达,倒置荧光显微镜和流式细胞术观察转染细胞上α-半乳糖基(α-Gal)的表达,流式细胞术检测人血清中IgM抗体和补体C3与转染细胞上α-Gal的结合。结果在BMI中发现了碱基长度为1116bp和1080bp的两个α1,3-GT基因剪接变异体,后者缺失了外显子5。pN-GT1或pN-GT2转染的A549细胞中,均检测到了α1,3-GT mRNA和α-Gal的表达,转染细胞膜上也检测到IgM和C3的沉积,且两种转染细胞中α-Gal的表达及IgM和C3的沉积没有差异(P>0.05)。结论成功克隆了BMI的α1,3-GT基因,并发现两种α1,3-GT基因剪接变异体。两种基因剪接变异体转染的细胞中,均检测到α1,3-GT mRNA的表达且α1,3-GT能催化合成具有生物学效应的α-Gal,这为BMI用于异种移植中涉及α1,3-GT基因操作的研究提供了基因背景资料。

Cloning of splicing variants of alpha1,3-galactosyltransferase cDNA of Chinese Banna Minipig inbred line and its expression in human cells

Objevtive To study the transfection and expression of the splicing variants of α1,3-galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line(BMI) in human A549 cells.Methods Full length of α1,3-GT gene cDNA was amplified by RT-PCR from total RNA of BMI liver tissue and cloned into T-A cloning vector.Two different splicing variants of BMI α1,3-GT cDNA were confirmed by sequencing 15 positive clones and inserted respectively into pEGFP-N1 to construct eukaryotic expression vectors pN-GT1 and pN-GT2.The vectors were transfected into human lung adenocacinoma A549 cells and the expression of α1,3-GT gene was detected by RT-PCR.The expression of the α-Gal epitopes on transfected cells was confirmed under fluorescent microscope and by flow cytometry using FITC-BS-IB4 lectin.The binding of IgM and complement C3 in human serum to α-Gal on transfected cells were measured by flow cytometry using FITC-anti-IgM and FITC-anti-C3.Results There was no other splicing variants of α1,3-GT cDNA found in BMI except GT1 and GT2,which were 1116 bp and 1080 bp in length respectively,the latter lacks exon 5.The expression of BMI α1,3-GT mRNA and the synthesis of α-Gal on A549 cells transfected with either pN-GT1 or pN-GT2 were detected,and the binding of IgM nature antibodies and complements C3 in human serum on transfected A549 cells were observed.The expression level of α-Gal and the deposits of IgM and C3 on transffected cells showed no significant difference between pNGT1 and pN-GT2.Conclusion The splicing variants of α1,3-GT cDNA of BMI could express in human cells,which provide the basis for genetic manipulation of the α1,3-GT of BMI for future xenotransplantation studies.