Studies On Heterogeneous Expression Of Goldfish Growth Hormone Ⅰ And Keratinocyte Growth Factor

(1) Fish growth hormone(fGH) produced by the pituitary cells of teleosts.It can increase the expression of protein,degradation of lipid and play an important part in the growth of fish.However,the content of native fGH in pituitary is extremely low and the expenditure of purification is too high.Fortunately,it is possible to produce recombinant fGH abundantly.And they have been proved in many cases the same function as the native ones without any side effect to human.Therefore,it is valuable to investigate the production of recombinant fGH.The gfGHâ… cDNA was amplified by PCR use the oligonucleotides which were designed to generate a BamHâ… site at 5' end and a Notâ… at the 3' end.The PCR product was purified with PCR purification kit and digested with BamHâ… and Notâ… digestion,used gel to harvest.It was then ligated into the corresponding and BamHâ… and Notâ… restriction sites in the expression vector pET28a and pGEX-4T-2,which was named pET28a-gfGHâ… and pGEX-4T-2- gfGHâ… .The recombinant expression plasmid pET28a-gfGHâ… was transformed into BL21(DE3) and fusion protein(His)₆ tag-gfGHâ… (about 26 KD) containing a(His)₆ tag at the N-terminus was expressed after 1 mM IPTG induction.MS identify and Western blotting indicated that the expressed recombinant protein was(His)₆ tag-gfGH.The recombinant protein was mainly in the form of inclusion body.The expressed product is purified with continuous ultrasonic wave so that we obtain the purified fusion protein of recombinant(His)₆ tag-gfGHâ… ,The high purified protein was used as antigen to immune mouse directly and high quality antiserum was obtained and the efficiency of antibody was 1:1600,which showed that the fusion protein possesses good immunogenicity.The vector pGEX-4T-2- gfGHâ… was transformed into BL21(DE3) which can be induced by 1 mM IPTG to produce a special protein GSTtag -gfGHâ… of 46 KD in molecular weight.The special protein can be mainly in form of soluble protein or inclusion body when induced at 28-37℃respectively.The cells induced at 28℃ were digested by lysozyme and disrupted by sonication.After being centrifuged the supernatant was purified by GST Resin.GSTtag-gfGHâ… fusion protein could be absorbed specifically on this column.The high purified GSTtag-gfGHâ… was dissolved in the sterile saline and was injected fancy carp(Cryprinus carpiod) intraperitoneally at the dose of 0.1μg·g^-1 body weight week^-1for one tested group,and control group was treated with sterile saline without gfGHâ… .After 4 week injections,the growth rate of fancy carp injected with purified GSTtag-gfGHâ… ,was found to be 24.68%heavier than the control,and 11.82%longer than the control. The analysis of test indicated GSTtag-gfGHâ… showed the significant growth-promoting effects in body weight and body length on fancy carp(P＜0.05) with significant difference.One of the hottest studies in plant genetic engineering is that using the plant bioreactor to product the useful medicine or antibody.Compared with the microbe like E coll.,plant as a bioreactor is edible,cost low,and has eukaryotic post-translational modifications.We constructed plant expression vector p1301-35S -gfGHâ… -Tnos,Then,the gfGHâ… gene was integrated into the genome of rice via Agrobacterium tumefaciens EHA105 mediation and rice effective transformation system respectively.Results indicated that the gfGHâ… has been transferred into rice cultivars(Zhong hua11) with PCR analysis,and GUS gene expression detection. The mRNA transcription of gfGHâ… in regenerated plant were detected by RT-PCR, the results showed that gfGHâ… can transcript in the transgenic rice,this indicates that gfGHâ… can be expressed stably.With PCR and RT-PCR analysis of transgenic T1 generation plant,We found that the heredity of gfGHâ… can succeed in giving transgenic T1 generation plant and gfGHâ… can transcript in the transgenic T1 generation plant,too.(2) Keratinocyte growth factor is a member of the fibroblast growth factor(FGF) family and was originally isolated from the conditioned medium of a human embryonic lung fibroblast cell line.KGF played a central role in the growth and renew of epidermal cell.It can accelerate the bladder,nephridium,intestines and corneal epidermal wound healing.Also,it can alleviate the chemotherapy and irradiation induced injure.Pichia pastoris bears characteristics that suit demands of producing recombinant biopharmaceuticals,such as the simplicity of gene manipulation, the ability of producing foreign protein at high level and performing many eukaryotic post-translational modifications.KGF gene fragment obtaintined by digestion PMD18-T-KGF with EcoRâ… and Notâ… directly inserted into pPIC9K plasmid to produce pPIC9K-KGF.Then the pPIC9K-KGF plasmid was linearized and transformed into P.pastoris by chemical transformation.The recombinant strains were selected by G418-MD plates,and then were identified Colony PCR,total genome DNA PCR.And the His⁺Mut⁺ phenotype was performed by MM/MD plates.The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE,The results showed that the KGF was detected by RT-PCR in the P.pastoris and a new protein band was found in SDS-PAGE with molecular mass of about 23 KD.These indicated that the KGF was correctly transcribed and translated in the P.pastoris.The influence of different factors on recombinant protein production during induction phase was studied.After 4 days induction in shake flask,the expression of protein reached maximum.