| Stem cell factor (SCF) and its ligand type III tyrosine kinase receptor are closely involved in the regulation of a wide range of tissue at different stages of life. They play an important role in various biologic phases,such as hematopoiesis, reproduction and regeneration. Human SCF is encoded by a gene located on chromosome 12q22-24,which including 8 exon. The gene is alternatively spliced at exon 6 encoding two isoform of SCF: soluble and transmembrane forms of SCF, and both of them have the same biological active. Soluble SCF is generated from SCF248 which is composed of 248 amino acids by spliced at a specific proteolytic cleavage site at Ala165. Soluble SCF sometimes exists as homedimer by noncovalent. It promotes viability and induced the proliferation of haemopoietic progenitor cells. SCF is potently synergistic when combined with growth factor such as: EPO> GM-CSF, G-CSF, IL-2, IL-3, IL-11, TPO. There are 3 clincal areas where SCF has been used today: (1)in vivo and ex vivo expension of stem cell, (2)stem cell mobilization, (3)therapetic of aplastic anaemia. It was claimed that rhSCF produced from E.coli has the same biological active as the natural glycoprotein of soluble hSCF.It is difficult to reach a high expression of rhSCF in E.coli. We have been studying the expression of rhSCF in E.coli since 1992. Though we have not got a high expressing bacteria, we have got lots of experience. In order to find the reason for rhSCF could not high express in E.coli, we analysed the result gained before with the help of software on computer.lt was pBV-220 vector that we selected to express rhSCF in DH5 a becteria. pBV-220 vector is constructed for high expressing gene in E.coli by the scientist in China. This vector is temperature sensitive. It has been used to express many genetic medicine and poly peptide, and often gave high expression. There are 3 factor that play an important role in high expression of pBV-220: (1) length of SD sequence, (2)RNA secondary structure of the sequence around ATG andTAA, (3) condon adaptive. We analysed the work done before, and draw the conclusion like this: both SD sequence and RNA secondary structure have little effect on high expression of rhSCF. Soluble SCF is composed of 164 amino acids. There are 57 amino acids whose condon adaptive is lower than 0.1, and it is 34.8 percent in totle amino acids. Also amino acids with lower condon adaptive often connected side by side existing here and there in the gene of rhSCF. So we concluded that condon adaptive maybe the reason for low expression of rhSCF.To highly express hSCF in bacterial strain,the gene of hSCF was corrected and primer was designed with the help of Goldkey software. The gene was synthesized, ligated by PCR in one tube, and cloned into a prokaryotic temperature sensitive vector pBV-220. Sequence analysed showed the insert gene has the same sequence as we designed After induction the recombinant bacteria could produce rhSCF about 27 percent of all bacterial protein. Western-blot showed a stained band at 19000 Da. The biologic assay showed that rhSCF could stimulate the proliferation of Mo7e cells. It is the amino acid codon adaptive that make SCF low expression.10 years before, researches on signal transduction of cytokine are developed in cell line for the limitation of the technology. With the development of CD34+ cell separating and culturing technology, it is possible to study the signal transduction of cytokine with the body cell in vitro. Erythropoiesis is clearly expatiated from 1998. Stem cell factor and its receptor have an important role in erythropoiesis. There are two main functions of stem cell factor in erythropoiesis. One is inhibiting apoptosis of CFU-E and BFU-E , the other is stimulating proliferation of CFU-E and BFU-E. So stem cell factor has its clinical usage in anaemia. We wonder if SCF has the same function on tumor cell as it is on the stem cell.The continuous cell line K562 is a human erythroleukemia line.lt could beinduced and show some specific marker of erythroid by so...
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