Study Of Generate Prosaptide NP14 By Gene Engineering

Prosaptide TX14 is a newly discovered neurotrophic factor. It is derived from the neurotrophic region of prosaposin.Both prosaposin and prosaptide TX14 can bind to the pertussis-toxin-sensitive G-protein, which activates extracellular-regulated kinases (ERKs),and stimulate neurite outgrowth and choline acetyltransferase activity in neural cells.Just because prosaposin is a newly identified neurotrophic factor and its wide distribution suggests that ProsaptideTX14 may have some other bioactivities beside its neurotrophic functions. So further study of its bioactivity and the possible molecular mechanism will make a foundation to its potent value of clinical usage.Genetic technology is an effective method to generate small peptide, we are going to synthesis gene NP14 according to the amino acid sequence of ProsaptideTX!4, and ligate each NP14 into multi-copies, then generate ProsaptideNP14 effectively in E.coli to study its bioactivity. The main contents of the study illustrate as following. AIM:Generate prosaptide NP14 by gene engineering METHODS:1. The gene fragment NP14 was derived from ProsaptideTX14, the first amino acid of ProsaptideTX14 was changed from T to S.Then, the NP14 fragment was ligated to 2NP fragment by PCR, and Met was added between each NP14 fragments to establish the protein lysis site.2. The pUC 18 vector was dephosphorylated by calf intestine phosphatase, andligated with 2NP gene fragment,thus the amplifasive vector(pUCl 8-2NP) was constructed through blunt ligation.3. Through blue and white screen, we got recombined vectorpUCl 8-2NP, and the positive clony was propagated in E.coli JM109, then extracted, purified and digested by EcoT14I, and was confirmed to contain 2NP fragment by sequencing.4. A great deal of 2NP fragments was produced after pUC 18-2NP was digested by EcoT14I, These fragments were ligated into multi-NP fragments through self-ligation.5. Multi-NP fragments were subcloned into pETEcoT, a vector derived from pET32a (+). A unique EcoT 141 (CCAAGG) cloning site was introduced into the modified pET32a(+) to allow unidirectoinal insertion of multiple coding sequences. Multiple copies of target gene were cloned as tandem repeats interspersing with two different sites, where the fusion protein could be cleaved with CNBr to release monomer peptide units remaining no any additional amino acid.6. Screen the clony through a vriaty of methods, and got multi-NP by PCR-array at last.7. The multi-NP fragments fused with trxA had been expressed as fusion protein. The different copies of fusion protein were expressed at high level in E.coli BL21 (DE3).RESULT:We synthesized 2NP fragment according to prosaptide, succeeded in constructing the amplifasive vector (pUC18-2NP) through blunt ligation and expressive vector (pETEcoT+8NP) through self-ligation and PCR-array. The multi-NP gene was expressed in E.coli after sequencing. Our work builds a solid foundation for further study on its neurotrophic bioactivity. CONCLUSION:Prosaptide NP14 can be self-ligated into multiNP and highly expressed in E.coli.