Human soluble CD40L cDNA Cloning and Expression of.docVIP

 PAGE \* MERGEFORMAT 11 Human soluble CD40L cDNA Cloning and Expression of [Keywords:] Prokaryotic expression Cloning of human sCD40L cDNA and construction of vector for expression in E. coli [Abstract] AIM: To clone human sCD40L (184-831) cDNA and construct a prokaryotic vector for expression in E. coli. METHODS: The human sCD40L (184-831) cDNA was amplified with the total RNA from the human tonsil by reverse transcriptase (RT) PCR, and was analyzed by enzymatic digestion and sequencing. Then, the insert of human sCD40L (184-831) cDNA was cloned into the vector pET28a for expression in BL21, and then the interest protein was identified by Western blot. RESULTS: The result of sequencing was accordant with the one reported. The prokaryotic expression vector was constructed successfully and the interest protein was expressed in BL21. CONCLUSION: The human sCD40L (184-831) cDNA has been successfully cloned and a prokaryotic system been constructed for expression. The interest protein was expressed successfully in BL21. [Keywords] CD40L; cloning; prokaryotic expression [Abstract] Objective: Using gene technology cloned extracellular region of CD40L molecules 184 ~ 831 fragment cDNA, to build its prokaryotic expression vector in order to establish the prokaryotic expression system. Methods: The method used RTPCR of total RNA from human tonsil was amplified CD40L molecules 184 ~ 831 fragment of the cDNA, in order to restriction enzyme digestion and DNA sequencing were identified, and the purpose of fragment was cloned into prokaryotic expression vector pET28a, so that expression of recombinant plasmids in BL21, using Western blot identification of the target protein. Results: Molecular cloning of a human sCD40L 184 ~ 831 fragment of the cDNA, DNA sequencing is fully consistent with the reported results; construct the prokaryotic expression vector fragment, and the target protein was expressed in BL21. Conclusion: The successful cloning of human sCD40L molecules