[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].
This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-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 alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA 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 alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.
Zhu
Shengming
S
Laboratory of Molecular Diagnosis of Cancer, West China Hospital, Sichuan University, Chengdu 610041, China.
Wang
Yanping
Y
Zheng
Hong
H
Cheng
Jingqiu
J
Lu
Yanrong
Y
Zeng
Yangzhi
Y
Wang
Yu
Y
Wang
Zhu
Z
D000818:Animals; D017849:Animals, Inbred Strains; D001483:Base Sequence; D002681:China; D003001:Cloning, Molecular; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013552:Swine; D013556:Swine, Miniature / Q000235:genetics*; D014162:Transfection
D011994:Recombinant Proteins; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine