Cloning And Expression And The Analysis Of The Biologic Activity Of Canine Epidermal Growth Factor

Epidermal growth factor (EGF) is a polypeptide which can promote theproliferation, differentiation and migration of cells. EGF participates in a series ofphysiological process such as embryonic development, tissue regeneration, endocrine,wound healing, gastric secretion and so on. In addition, it pays an important role intransduction events of cells, and its receptor has important relationship with tumorgeneration. Recently, the human epidermal growth factor has been used in medical andcosmetic areas widely. The research and development of canine epidermal growthfactor is very important to canine health care.1. Cloning and sequence analysis of genes encoding canine epidermal growthfactorTo clone and analyze the sequence of canine epidermal growth factor (cEGF) genefragments from different varieties of canines,and analyze difference comparisonamong the cEGF and the EGF of several common species. Eleven canine bloodsamples were collected from Pet hospital.The exon20and exon21of Canis lupusfamiliaris chromosome32, Coding cEGF gene N-terminal and C-terminal structuredomain respectively, were amplified by PCR from the DNA extractes of the bloodsamples, After purification，the gene fragments were ligated with plasmid pMDTM18-T Vector to construct recombinant plasmids，and transformed into E.coli DH5αrespectively.Positive bacterial clones were identified by PCR methods.The sequencesof inserted cEGF fragments，concatenation of texon20and exon21, were determinedand analyzed by BLAST and DNAStar software. Results The amplified cEGF genefragments of the eleven canine were about156bp in length, Encoding52amino acids,The results of pair comparison between the different fragments the general homologywas between97.4%-100%. Amino acid sequences homology, According to the cEGFgene encoding the deduced, was between98.1%-100%，all amino acid sequence wereconsistent except the Doberman Pinscher EGF amino acid sequence by the the Rmutation for F in In fifty-second site. The homology was94.2%between cEGF and Felis catus EGF. Conclusion Different varieties of canines EGF genes were veryconservative, the Homology between cEGF and Felis catus EGF was huge.2. Prokaryotic expression, purification and bioactivity identification ofrecombinant canine epidermal growth factor and preparation of the polyclonalantibodies anti-cEGFThe cEGF genes were synthesized and cloned into prokaryotic expression vectorby DNA recombination technology, and the recombined plasmids were transfected intoE.coli expression system to obtain a high production of cEGF, the biologic activity ofcEGF was also detected. In details,4primers were designed, the cEGF gene wasamplified by SOE-PCR, the the obtained fragments were cloned into pET-24a andpET-28a respectively. cEGF were expressed by E.coli expression system and purifiedsubsequently, the biological activity was assessed by MTT assay, and the affection ofthe tag proteins to cEGF was detected too. Immunized Japan big-ear white rabbit usingthe purified cEGF, get polyclonal antibodies anti cEGF. Detect the titer of polyclonalantibodies through indirect ELISA. The length of cEGF gene amplified fromSOE-PCR was156bp, encoding52amino acids.3recombined plasmids wereestablished, encoding61,75and95aa respectively. The expressed cEGF proteinsinduced by IPTG were mainly inclusion bodies. Denaturalization, purification andrenaturation progress were performed gradually.52mg rcEGF1,16.1mg rcEGF2and93mg rcEGF3were obtained from respective1L bacterium liquid. rcEGF1, rcEGF2and rcEGF3showed gradually decreased biological activity. the titer of rabbit serum is1:512000，The results suggested that6x His tag-recombined cEGF shows the highestbiological activity and the molecule weight of the tags can affect the biological activityof the recombined proteins.3. Expression of canine epidermal growth factor in pichia pastoriscEGF fragment was inserted into seeretory pPICZαA pichia pastoris expressionvectors. The resulted plasmid was electroporated to pasteur and summarized yeast(X33) to express the recombinant protein after the induction of methyl alcohol.Expression product was detected by SDS-PAGE and western blot. The result showedthat the recombinant protein were secretory expressed.