Cloning Of Pro-β-Nerve Growth Factor Gene Using A-T Cloning Method And Expression In Pichia Pastoris Stain

ObjectiveNGF was one of the nerval trophic factors, it played an important role in the survival, growth, development, differentiation, repair and regeneration of nerve cells. Meanwhile, NGF was also thought to have a profound effect on im-munoreaction.NGF has being had more and more effects on many kinds of diseases, such as Alzheimer's and Parkinsonian's disease. The recent studies demonstrated that NGF also had some effects on such diseases as peripheral nerve impairment, optic nerve disease, brain trauma and so on.The amount of NGF was less in nature. So, it was imperative to obtain an amount of active NGF by the gene - recombined technology.The present study was designed to obtain the pro - - NGF gene by PCR amplification, the recombinant pGEM -T -NGF by subcloning, the active - NGF protein by inserting the gene into the expression vector pPIC9K and transforming into the Pichia pastoris stain cells GS115.Methods(1) The pro - (3 - NGF gene was amplified by using PCR with a pair of specific primers designed in according to the - NGF gene sequence and Taq plus pfu DNA polymerase.(2) The recombined plasmid pGEM - T - NGF was obtained by A - T cloning method. PCR product was ligated directly with pGEM - T vector to be formed the recombinant pGEM - T - NGF and the mix was transformed into the competent E. coli cells (JM109) that was done by Calcium Chloride method.We plated the transforming mix onto LB plates with Ampicillin/ X - gal/IPTG. The transformations were analyzed by Blue - White Spot Assay. The white spots were detected by enzymolysis, PCR and sequencing respectively.(3) The recombinant pGEM - TfJ - NGF and expression vector pPIC9K were digested doubly with SnaB I and Avr H respectively. The aimed DNA was isolated by 1.2% argarose gel electrophoresis, and purificated by Gel DNA Abstraction Kit. The p - NGF was ligated with pPIC9K by T4DNA ligase and was transformed into competent E. coli cells JM109.(4) The positive colonies from the transformed cells growing on the LB/ Amp plate were picked into LB medium with Amp, and the recombinant pPIC9K- - NGF was abstracted using Plasmid DNA Mimiprep Kit, and its sequencing was performed with primers -factor and 3'AOX1.(5) Both pPIC9K - NGF and the parent vector pPIC9K was linearized by Sacl. Then, they were transformed into Pichia pastoris strain, GS115, by spher-oplasting. The spheroplast was spread onto the His- RDB plates. The positive colonies were obtained from the His ~ RDB plates and spread onto YPD plates. To identify the Mut+ /Mut" phenotype, positive colonies were pickde and acted as templates. The PCR was performed with the primers 3'AOXl and 5'AOXl.(6) The recombinant strain was inoculated with RDB medium at 30^Cwith shaking at 200rpm and induced by 100% methanol ( final concentration 0.5% ). The methanol was added every 24 hours. Supernatant at 24, 48, 72, 96 hours was obtained in order to be analyzed.(7) The expression supernatant was analyzed using SDS -PAGE electrophoresis and Western blotting with rabbit - anti - rat polyclone NGF antibody.Results(1) The 1. 2% agarose gel electrophoresis results of PCR showed the 748 bp gene fragment was obtained from abstraction of placenta DNA and amplification with a pair of special primers. The sequence of (3 - NGF gene obtained was the same as the reported.(2) In the Blue - White Spot Assay, the white colonies were positive ones that contained the recombinant. After the recombinant pGEM - Tp - NGF was purified and digested by Pvu II , the results of 1.2% agarose gel electrophoresis showed that the positive bands appeared in both 2564bp and 1185bp.(3) White colonies were acted as templates for PCR. Their PCR products were analyzed by 1.2% agarose gel electrophoresis, which revealed that the positive band appeared in 748bp. The recombinant pGEMT - P - NGF sequence was the same as the reported.(4) Both pGEM - Tp - NGF and pPIC9K contained SnaB I and AvrH restriction site. The recombinant pPIC9Kp - NGF was obtained throu...