The Construction Of Eukaryotic Expressing Vector And Transfection Of Human Epidermal Growth Factor Gene

Epidermal growth factor ,one member of growth factor famalily,is a mitogen of epithelial cell,epidermis cell,mesenchyma cell and other sensetive cells.lt is also one of the potent cytokines that can promote cells to proliferate and differentiate .It has been identified that human epidermal growth factor can stimulate tissues stemming from ectodem and endoderm to mitosis and synthesize protein,thus can promote the repairment of skin ,corneal and gastroenteric mucosal epithelium.At present,EGF recombinant protein has been studied in the healing of surgical wound ,corneal injury and gastro-duodenal ulcer.A part of them have been utilized in the clinical treatment.Recombinant protein of EGF is a low molecular weight protein with a short Tl/2 and it can be easily degraded .And the cost of production and purification is so high that it prevent its widely used.If hEGF gene is brought into wound by a vector,it will secrete recombinant protein in the local tissue and promote the process of wound healing.In this report, hEGF gene was cloned with gene engineering technique and eukaryotic expressing vector of hEGF was constructed.With the help of liposome , pcDNA3.1-hEGF was brought into cornea.This research will lay foundation for the gene therapy of wound. Methods 1. Construct the clone vector of hEGF geneThe total RNA was extracted from tissues which are abundant in hEGF. Reversetranscript reaction and PCR were used to obtain the DNA of hEGF .Then transfer hEGF gene into clone vector-PGEM-TEasy to mutiply. Select and identify the positive recombinant.2. Construct the eukaryotic expressing vector of hEGF and analyse the sequence of hEGF Restriction enzyme- Hind III. BamH I were used to remove hEGF gene fromPGEM-TEasy- hEGF and transfer it into eukaryotic expressing vector- pcDNA3.1.Biological software was used to analysize the sequence of hEGF.3. Gene transfectionLipofectin and pcDNA3.1-hEGF were mixed to transfect the corneal cell.PCR. RT-PCR and Immunohistochemistry were used to detect the expression of hEGF at three different levels: DNA > RNA , Protein. Results 1. Construct the clone vector of hEGF geneThe gene of hEGF was obtained by RT-PCR from fetus submaxillary and salivary glands ,rts base pair is 162. hEGF gene in the fetus submaxillary was combined with vector-PGEM-TEasy to construct PGEM-TEasy- hEGF.Blue and white scan experiment . enzyme reaction and PCR were used to select the positive clone. 2.Construct the eukaryotic expressing vector of hEGF and analyse the sequence of hEGFhEGF gene was transfered from PGEM-TEasy- hEGF into eukaryotic expressing vector-pcDNA3.1. The sequence analysis results show that base pair homology is 98.2% between hEGF gene and Genebank sequence.Amino acids are identical. 3.Gene transfectionIn the 21st day ,DNA and RNA of hEGF can be detected in the transfection tissues with PCR and RT-PCR.Immunohistochemistry is used to detect whether the protein can be expressed or not. hEGF protein can be detected in two weeks.After that, hEGF can not be detected by this method. Conclusions1 .The amount of hEGF is different in tissues and it is high in the fetus submaxillary gland which is associated with the function of hEGF to promote fetus development. 2.hEGF clone vector PGEM-TEasy- hEGF has been constructed. 3.The eukaryotic expressing vector of hEGF has been constructed.4.Pair homology is 98.2% between hEGF gene and Genebank sequence.Amino cides areidentical.S.Lipofectin can bring hEGF gene into cell and pcDNA3.1-hEGF can secrete the protein.