High Level Expression Of The Human’s Type Ⅲ Collagen Peptite In Streptococcus Thermophilus And Optimization Of Fermentation

Collagen is a class of extracellular stromatin. It’s the major protein of connective tissue, which plays an important role in maintaining the integrity of the various organizations. Collagen not only has a unique structure and is rich in diversity and has specificity of tissue distribution but also have a close relationship with the function of organ and body aging. But the production and use of human collagen exists ethics and moral issues. According to the human type III collagen’s three peptide functional sequence and the design principles of the synthetic biology, this research transforms the Streptococcus thermophiles by genetic engineering method, which avoides the problems of ethics and morality..This research manual designed, optimized and synthesised the gene of α1 chain of collagen type III(col III) by the method of the codon optimized. Then fragments were inserted into the linearized vector p SB1C3, constituted the connection plasmid(p SB1C3-col III-nis I). The fragment(col III) was cut down by the method of the double digestion method. Then col III was inserted into the carrier p MG36 e, constituted the recombinant plasmids(p MG36e-col III-nis I). The plasmids were transformed to Streptococcus thermophilus by electric shock and validated. The obtained engineering bacteria was verified though plasmid, PCR verification, SDS protein electrophoresis. The sequencing results were 99% similar compared with the target sequence.This research screened out the highest expression strain as engineering strain by 12% SDS-PAGE electrophoresis analysis. And the stability of the recombinant plasmids was detected. Using different crushing methods to disrupt cell. After centrifugation, the frozen supernatants, precipitation, and inclusion bodies were verified though SDS-PAGE electrophoresis, western-blot analysis, which detected the expression and location of recombinant proteins. Express the collagen peptide and purified it though a nickel column to obtain high purity collagen peptide.Based on economic principles and effective principle, this research optimized the medium’s carbon and nitrogen sources, temperature, erythromycin concentration, temperature, inoculation, erythromycin concentrations, and made the growth curve of the untransformed bacteria and the engineered bacteria to prove this study have no impact on Streptococcus thermophiles’ growth. This research found that broth OD600 value and protein concentrations have a positive correlation. The best ratio of the medium was 1% glucose, 2% peptone and 1% yeast extract. The bast culture condition was inoculating 0.4% bacteria in ELL medium containing 2.5mg/m L erythromycin, under 42℃ until OD600 was 1.3. The expression of collagen was 21.97% of the total protein.