Using The Shuttle Plasmid In The Synechococcus Sp.pcc7942 Heat Induced Expression The T¦Á <sub> 1 </ Sub>,

Cyanobacteria, or blue-green algae, are photoautotrophic organisms with many special properties, which make them very attractive for molecular biological studies. With the more studies on this field, producing better strains of cyanobacteria and genetic products through recombination DNA techniques are two important aims of cyanobacteria genetic engineering. And great progress has been made in these areas.The experiment began with the shutter vector pPRS- 1, in which endogenous small plasmid (1.5kb) of Plectonema boryanum was cloned as replication origins of cyanobacterium. The 5.8kb shuttle vector was reconstructed and a small vector pPUR(4. 19kb) was obtained, which preserved the 1.5kb plasmid. HindIII was used to digest pEUTMT1, and a 4.21kb fragment containing heat-shock promoter groESL~ target gene UB-Ta1~ rbcS polyA terminator and Klnt gene was obtained by electrophoresis. Then the fragment recombined with the two shutter vectors pPUR and pPRS-1, and obtained two shuttle expression plasmids, pPUREUT(8.4kb) and pPREUT(lO.O8kb).The recombinant plasmids were directly transformed into Synechococcus sp.PCC7942 respectively. The kanamycin-resistant transformants were obtained by kanamycin screening. Southern blotting analysis showed UB-Ta1 gene had been transformed into Synechococcus sp.PCC7942. Under time gradient in 420C, a special protein in transformed strains Which molecular was 1 lkDa was detected at 30mm. The protein was tested to the product of target gene UB-Ta1 by Western-blot. Gel-analysis showed that the expression content of target protein was at least 6.6% in the group protein of Synechococcus sp.PCC7942. The result of the stability of the shuttle plasmid in the transformed strains showed plasmid would be lost with the growth of the algae without selected factor. The contained ratio decreased from 75~-1OO% to 14.1-11.3% after cultivated continuously for 72h. But they can keep stable in kanamycin. Moreover, we found the transformed strains have the same biological function as Ta1. They can increase the T-cell sub-population of mice, and improve the ratio ofCD4ICD8.