Construction of eukaryotic expression vector with gene of human CD40L and its expression in plasma membrane of NIH3T3
AIM: To amplify and express human CD40L gene in the plasma membrane of NIH3T3 for an easy and effective method to produce human CD40L protein. METHODS: Human CD40L gene was amplified by gradient RT-PCR from Jurkat cell line and subcloned into the T vector. After sequencing, pcDNA3. 1-CD40L was constructed and transfected into NIH3T3 cell line. The expression of CD40L was tested by FACS. RESULTS: The CD40L gene was successfully amplified from Jurkat cell line. Sequencing identified a clone with CD40L gene without any muta-tion. After transfection into NIH3T3 and primary selection with G418, about 41% cells successfully transported the expressed CD40L protein to the plasma membrane. CONCLUSION: Human CD40L gene can be amplified from Jurkat cell line. pcDNA3.1 vector expresses CD40L successfully in NIH3T3 cell line and the expressed protein is partially transported to plasma membrane.
CD40 ligandreverse transcriptase polymerase chain reactionconstruction of PODNA3. 1-CD40LNIH3T3flow cytometry
NETL
NSTL
万方数据
维普
