Man Of Cd7 Cdna Cloning, Expression And Function

CD7 is a single-domain Ig superfamily molecule expressed on human T and NK cells, as well as on cells in the early stages of T, B, and myeloid cell differentiation. CD7 is highly expressed on malignant immature T cells and is generally absent on malignant mature T cells, such as CD4+ Sezary leukemia and HTLV-1+ adult T-cell leukemia cells. CD7 is also involved in HIV infection, rheumatoid arthritis.However, there are still a lot of unexpected field that urge us to investigate, so it is important to obtain sufficient quantity and high quality of CD7 protein and establish cell model that constitutively and stably express CD7. Based on this goal ,we conducted following experiments: 1 .Expression of CD7 extracellular cDNA in E.coli and purification of CD7-biotinylated fusion protein. (1) With PCR Technique ,we made CD7 coding sequence having the same open reading frame as biotin binding preotein , meanwhile defected the sequence encoding signal peptide of CD7 and added a terminator that E.coli favor.Then the sequence encoding extracellular CD7 was cloned into Prokaryotic expression vector Pinpointxa-3 to obtain positive recombinant plasmid named as Pinpointxa3-CD7. DNA sequence analysis confirmed the extracellular CD7 coding region fragment was the same open reading frame as the biotin fusion protein in the vector and the DNA encoded the same Aa sequence as the published CD7 extracellular Aa sequence (2) For the expression of recombinant fusion protein ,E.coli DIJ5 a was [V used as expression host .Under the induction of IPTG, an fusion protein was expressed in E.coli and could be detected by SDS-PAGE .7.3% of fusion protein from total bacterial protein were produced after the induction of 600mmol/L IPTG at 370C for 4h,but after 9h induction at 3O~C,the yield of the fusion protein was 13.48% of total bacterial protein. The molecular weight of the recombinant fusion protein was about 3OkD . Western-blotting indicated that this was the specific band of CD7-biotinylated fusion protein. Using avidin resin, we purified the CD7-bi髏inylated fusion protein. (3) The successful expression and purification of CD7-biotinylated fusion will provide sufficient materials for further studies on CD7. 2.The expression of CD7 cDNA in mammalian cells and function study: (1) Recombinant eukaryotic expression vector pclneo-CD7,pcDNA3-CD7 were constructed by cloning human CD7 eDNA containing the full length open reading frame into eukaryotic expression vector pclneo and pcDNA3 between the site of XhoL. XbaI. (2) HEK293 cell was transfected with pcDNA3 and recombinant eukaryotic expression vector pcDNA3-CD7 by electroportion technique, meanwhile Jurkat cell was transfected with pcDNA3-CD7, pcDNA3 by calcium phosphate-mediated gene transfer.The transfected cells was selected under the pressure of 0418 for two to three weeks. RT-PCR and FCM analyses showed that the 0418 resistance cells could express CD7 mRNA and CD7 protein. (3) Investigation for the the role of CD7 in IL-8 production induced by LPS: The response of HEK 293 and HEK 2931CD7 to LPS was observed by measueing IL-S .Both cell lines were stimulited with 1 Li g/ml LPS for 6h ,the supernant was collected for ELISA analysis. The results showed that CD7 had no significant effects on IL-8 production and suggest CD7 m...