Liu Mei, Zhu Sheng-Ming, Zheng Hong, Wang Yu, Wang Zhu, Yang Ya-Jun, Wu Yan-Xia, Zeng Yang-Zhi, Wang Yan-Ping
Laboratory of Molecular Diagnosis of Cancer and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2012 Mar;43(2):145-50.
To study the transfection and expression of the splicing variants of alpha1, 3-galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line (BMI) in human A549 cells.
Full length of alpha1,3-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 alpha1,3-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 alpha1,3-GT gene was detected by RT-PCR. The expression of the a-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 a-Gal on transfected cells were measured by flow cytometry using FITC-anti-IgM and FITC-anti-C3.
There was no other splicing variants of alpha1,3-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 alpha1,3-GT mRNA and the synthesis of a-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 alpha-Gal and the deposits of IgM and C3 on transffected cells showed no significant difference between pN-GT1 and pN-GT2.
The splicing variants of alpha1,3-GT cDNA of BMI could express in human cells, which provide the basis for genetic manipulation of the alpha1,3-GT of BMI for future xenotransplantation studies.
研究中国版纳微型猪近交系（BMI）α1,3-半乳糖基转移酶cDNA剪接变体在人A549细胞中的转染及表达情况。
从BMI肝脏组织总RNA中通过RT-PCR扩增α1,3-GT基因cDNA全长，并克隆至T-A克隆载体。对15个阳性克隆进行测序，确认了BMI α1,3-GT cDNA的两种不同剪接变体，分别插入pEGFP-N1构建真核表达载体pN-GT1和pN-GT2。将载体转染至人肺腺癌A549细胞，通过RT-PCR检测α1,3-GT基因的表达。利用FITC-BS-IB4凝集素在荧光显微镜下及通过流式细胞术确认转染细胞上α-Gal表位的表达。使用FITC-抗IgM和FITC-抗C3通过流式细胞术检测人血清中IgM和补体C3与转染细胞上α-Gal的结合情况。
在BMI中未发现α1,3-GT cDNA的其他剪接变体，除了GT1和GT2，其长度分别为1116 bp和1080 bp，后者缺少外显子5。检测了用pN-GT1或pN-GT2转染的A549细胞上BMI α1,3-GT mRNA的表达及α-Gal的合成情况，并观察了人血清中IgM天然抗体和补体C3在转染的A549细胞上的结合情况。pN-GT1和pN-GT2之间，转染细胞上α-Gal的表达水平以及IgM和C3的沉积无显著差异。
BMI的α1,3-GT cDNA剪接变体可在人细胞中表达，为未来异种移植研究中对BMI的α1,3-GT进行基因操作提供了依据。
