中国近交系版纳微型猪α1，3-半乳糖基转移酶基因的克隆及其真核表达载体的构建

Cloning of Chinese Banna Minipig Inbred-line α1,3-Galactosyltransferase Gene and Construction of Its Recombinant Eukaryotic Expression Vector

克隆中国近交系版纳微型猪(Banna minipig inbred-line,BMI)α1,3-半乳糖基转移酶基因(α1,3-galacto-syltransferaseα1,3-GT)并构建其真核表达载体。从版纳微型猪肝脏中提取总RNA,RT-PCR扩增出α1,3-GT cD-NA全长序列,克隆到pMD18-T载体,测序后再克隆到质粒pEGFP-N1中,构建α1,3-GT真核表达载体pEGFP-N1-GT。用脂质体法瞬时转染人肺腺癌细胞A549,RT-PCR检测转染细胞中α1,3-GT mRNA的表达;利用荧光素标记的植物凝集素(FITC-BS-IB4lectin),直接免疫荧光法检测转染细胞中α1,3-GT合成α-Gal表位的能力。结果显示版纳微型猪α1,3-GTcDNA全长1116bp,其序列与GenBank中小鼠和牛α1,3-GTcDNA具有高度同源性,与猪α1,3-GTcDNA全长序列完全一致。酶切和PCR证实重组真核表达载体pEGFP-N1-GT构建成功。转染pEG-FP-N1-GT的A549细胞有α1,3-GTmRNA表达,在其细胞膜检测到α-Gal表位的表达。中国近交系版纳微型猪α1,3-GTcDNA的克隆及其真核表达载体的构建为该基因在异种器官移植和肿瘤免疫治疗中的进一步研究和应用奠定了基础。

This study sought to clone Chinese Banna minipig inbred-line（BMI） α1,3-galactosyltransferase （α1,3- GT）gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length eDNA of a 1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, α1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of α1,3-GT mRNA in transfeeted cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method ,which was performed to observe the α-Gal synthesis function of BMI α1, 3-GT in transfected cells. The results showed that full length of BMI α1,3-GT eDNA was 1 116 bp. BMI α1,3-OT eDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of α1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of α-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI α1,3-GT eDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI α1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.