Construction of expression vector from different transcripts of RHD gene.
- Author:
Jun-Jie XIE
1
;
Yan-Ling YING
;
Xian-Guo XU
;
Fa-Ming ZHU
;
Chao-Peng SHAO
Author Information
1. Dalian Medical University Postgraduate School, Dalian, Liaoning Province, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Gene Expression;
Genetic Vectors;
Humans;
Molecular Sequence Data;
RNA, Messenger;
genetics;
Rh-Hr Blood-Group System;
genetics;
Sequence Analysis, DNA
- From:
Journal of Experimental Hematology
2012;20(1):173-177
- CountryChina
- Language:Chinese
-
Abstract:
RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh(D) positive umbilical blood cells of newborn. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/V5-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD gene, which lays a foundation for further exploring the membranous protein expression of Rh(D) antigen.
