Study On High Expression Levels Of Human Nerve Growth Factor Beta In Milk Of Goats By Adenoviral Vectors

NGF 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 immunoreaction.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 NGF-βgene by PCR amplification,the recombinant pShuttle- NGF-βby subcloning,and then RIES-GFP gene was inserted into pShuttle- NGF-βby enzyme. The resultant plasmid was linearized by enzyme and subsequently contransformed into BJ 5183 with the adenoviral backbone plasmid pAdEasy-1. Recombinants were confirmed by restriction endonuclease analyses. The adenoviruses were packaged and amplified in 293 cells and the viral tite was check by GFP. Then the adenoviruses were injected into the mammary gland to infect the goat mammary glandular epithelial cells. A plenty of the active hNGF-βprotein was obtain by Mammary gland bioreactor of goats.The results showed:1. The 1.0% agarose gel electrophoresis results of RT-PCR showed the760 bp gene fragment was obtained from human embryonic liver RNA and amplification with a pair of special primers. The sequence of NGF-βgene obtained was the same as the reported.2 After pShuttle-hNGF -β- IRES-GFP which was digested and identified by EcorⅠ, NotⅠa nd SphⅠw as right, the correct plasmid was linearized by PmeⅠand subsequently contransformed into BJ 5183 with the adenoviral backbone plasmid pAdEasy-1. The 1.0% agarose gel electrophoresis results of recombinant adenovirus plasmid Ad-hNGF-βdigested by restrictive endonuclease PacⅠwas 2.9kb or 4.5kb special band. It confirmed that the Ad-hNGF-βwas constructed successfully. The homologous recombination in bacteria was a convenient and efficient way to construction of recombinant adenoviral vectors. 3 Ad-hNGF-βvecter was transfected into HEK293 cell by liposome. After the fluorescence was observed in 293 cells which were infected, the viral supernatant was collected. Western blot showed that hNGF-βprotein was expressed in supernatant.It confirmed that Ad-hNGF-βwas expressed effectively in vitro.4 The packaged recombinant adenoviruses was transducted into mammary gland epithelium of goat. After 3 days, Western blot showed that hNGF-βprotein was expressed in collected viral supernatant at levels up to 4.1mg/L. It revealed high level of hNGF-βprotein was expressed in mammary gland epithelium of goat.5 High expression level hNGF-βwas obtained in milk of goats by the test of infecting mammary gland epithelia with recombinant adenoviruses. The highest expression level of hNGF-βattained 196.8mg/L. It confirmed the feasibility of expressing heterologous protein in mammary gland mediated by recombinant adenovirus as vector.6 It was Adenoviral re-administration that hNGF-βwas not detected in the milk of any of the animals re-infused with the Ad-hNGF-βvector regardless of the same or double quantity of viral vectors. It showed that the cellular and humoral immune response might have contributed to preventing a re-infection of the virus.