Construction Of An Engineering Bacteria Strain Containing N～α-desacetylthymosin α1 Gene Sequence, And Analysis Of The Expressed Protein Of Tα1

Thymosin α1 (Tα1) is an acidic peptide which contains 28 animo acids without including Met, Cys or amino acid residues. This peptide doesn't contain disulfide bond or glycosylation. The only modification of Tα1 peptide is acetylation in N- terminus.Here a unique secretion expression technology in E. coli was used. Recombinant plasmid containing Tα1 gene was transferred in to E coli, and the expressed protein was secreted directly into cytoplasm. So the downstream purification was simplified, and the production cost was decreased. Both and natural Tα1 have the same amino acid composition, but Tα1 expressed from E coli is without acetylation in N- terminus. In 1998, scientists in America have studied in vitro biological activity of Tα1 , and the results showed that acetylation in N-terminus didn't influence the biological activity of Tα1.In order to obtain a high yield expression of Tα1, a successfully construction of genetic engineering plasmid (PST II- Tα1) for expression is the critical step. We took PST II as the primary plasmid, which consist of alkaline phosphatase promoter (PhoA promoter), translation enhancer sequence, Shine-Dalgano sequence, ST II sequence, Amp and Tet resistance gene, and replication origin. After synthesizing aim gene of Tα1, the gene was cloned into downstream promoter of PST II. Tα1 cDNA was synthesized by step-by-step chemistry method. After four of the double complementary oligonucleotide fragments were synthesized and paired, the cohesive ends containing enzyme cleavage sites for endonucleases of Hind III and SphI were formed. The recombinant plamid was constructed by double-enzyme cleavage method, and the recombinant plasmid (PST II- Tα1) with aim gene fragment was obtained by connection, transfer, idenfication and screening, etc. The recombinant plasmid structure was verified by double-enzyme cleavage and gene sequence analysis. In order to obtain Tα1 which we need, selection of a approprite genetic engineering expression bacteria for gene expression is very important. Here, competent cells of fresh E Coli bacteria (W3110) were prepared by Cacl2 method, and the genetic recombinant plamids pTα1which were identified were transferred in to W3110 cells. The genetic engineering expression bacteria strain (pTα1/W3110) was obtained by plate culturing method. The plasmids were isolated and purified after enlargement culturing, and verified by double-enzyme cleavage method. An experiment of induced expression was carried out with engineering bacteria (pTα1/W3110). The engineering bacteria strain which had a highly expression yield was selected by SDS-PAGE so as to establish the primary seeding stock , and a sets of identifications for expressing protein were studied. Using SDS-PAGE method, Compared with total protein expressed from blank bacteria as control, a new protein band presented at position of 3000 Da. in expression protein from engineering bacteria with pTα1. and its molecular weight is identical to that of Zadaxin as a contrast., demonstrating the expression of Tα1 protein. Accurate molecular weight of this band was 3066 Da. determined by mass spectrum assay, and it is identical to the theoretical molecular weight of Tα1. Furthermore, amino acid sequence of the above expressing protein was exactly correct by whole sequence amino acid analysis. Also T cell activity assay-E rosette formation assay demonstrated that expression protein had the similar biological activity as compared with thymosin synthesized by chemistry method.In summary, we have successfully constructed genetic engineering bacteria (pTα1/W3110) with secretary expressing character. The bacteria strain can highly express a single protein product of Tα1. Successfully construction of this engineering bacteria strain has established the basis for further downstream purification of Tα1 product. We will continue this research work so as to obtain Tα1 product with higher purity and higher biological activity.