Cloning, Sequencing and Preliminary Expression of Human RP2 Gene.
RP2 is an X-linked retinitis pigmentosa gene, which was newly discovered by positional cloning. A polymerase chain reaction (PCR) was conducted to screen a full-length cDNA fragment, defined as hRP2a, which included the coding region of hRP2, in a human retina cDNA library. HRP2a gene was cloned into the pJLA503 vector and hRP2 gene was subcloned into the expression vector pP(RO)EX HTa. Polymorphism was demonstrated at two sites through DNA sequencing. The recombinant pP(RO)RP2 was transformed into Escherichia coli strain DH5alpha and the expression of a 6xHis tagged hRP2 fusion protein was induced by IPTG. Band density scanning of stained gel was performed to estimate the percentage of the recombinant protein in the total bacterial protein. The ratio was 7% when the expression was induced at 30 degrees and was 5.6 % at 37 degrees. The cloning and expression of hRP2 gene in E.coli established a basis for the further purification and studies of RP2 for its physiochemical identity, immunohistochemistry and structure-function relationship.
Liu
Xiao-Jun
XJ
Human Genome Lab, Institute of Genetics, Fudan University, Shanghai 200433, China. jhchai@fudan.edu.cn
Chai
Jian-Hua
JH