Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector.
- Author:
Shengming ZHU
1
;
Yanping WANG
;
Hong ZHENG
;
Jingqiu CHENG
;
Yanrong LU
;
Yangzhi ZENG
;
Yu WANG
;
Zhu WANG
Author Information
1. Laboratory of Molecular Diagnosis of Cancer, West China Hospital, Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Animals, Inbred Strains;
Base Sequence;
China;
Cloning, Molecular;
Galactosyltransferases;
genetics;
metabolism;
Genetic Vectors;
genetics;
Molecular Sequence Data;
Recombinant Proteins;
genetics;
metabolism;
Sequence Analysis, DNA;
Swine;
Swine, Miniature;
genetics;
Transfection
- From:
Journal of Biomedical Engineering
2009;26(2):360-365
- CountryChina
- Language:Chinese
-
Abstract:
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.
