Expression Of Human Growth Hormone Gene In Antheraea Pernyi Pupa By Using Antheraea Pernyi Nucleopolyhedrovirus Expression Vector System

Antheraea pernyi nucleopolyhedrovirus (ApNPV) is a species of the insect baculovirus that can infect the tussah pupa with high selectivity. The expression vector system has been developed based on this selective infecting characterist of ApNPV and successfully applied to express the green fluorescent protein gene in tussah pupa. While, there is no literature reported the application of recombinant ApNPV to express protein with biologic activity.This study reported that human growth hormone(hgh) gene was expressed in Antheraea pernyi pupa by using ApNPV. The transfer vector, pApM748BE/hgh, was constructed and cotransfected into Pc-01 cell with the genomic DNA of ApNPV-Δph/egfp+. The recombinant virus, ApNPV-Δph/Δegfp/hgh+, was selected with the limited dilution method and identified by using PCR method. Pupae of Antheraea pernyi were infected by ApNPV-Δph/Δegfp/hgh+ and the expression of hgh was analyzed by using Western-blot and ELISA. The results showed that hgh was obviously expressed in pupae at 7 dpi(days post infection). Contents of the recombinant hGH(rhGH) in haemolymph of pupae were maintained at higher level at 13 to 14 dpi, but relatively unstable at 15 to 20 dpi. The highest content of rhGH in haemolymph of pupae was 251.18μ.g/mL as shown by ELISA quantitative analysis in comparison with hGH standard sample. The rhGH from pupae could stimulate the proliferation and colony formation of K562 human erythroleukemia cells by in vitro assay, which demonstrated that the rhGH from pupae of Antheraea pernyi was active with hGH. The separation and purification method of recombinant hGH from Tussah pupa which infected by recombinant ApNPV was verified by affiity chromatography.In this job, it is promising to widen the using field of hGH, and develop drugs and functional foods based on rhGH preparation from Antheraea pernyi Pupa.