The Recombination And Heterogeneous Expression Of An Anti-tumor Gene In The Chloroplast Of Chlamydomonas Reinhardtii

Our lab had cloned allophycocyanin gene (apc) from ayanobactreia in 1998. The gene was ligased in plasmid PET28a+, and the allophycocyanin (APC) was expressed in E. coli with a high level. The purification protein can inhibit the S180 and liver tumor in mice from 40-60%. But using E. coli to product the protein had to suffer the purification and the refolding of inclusion bodies. Because both ig and ip can inhibit tumor remarkable, which promote us to exploit a new mucosal anti-tumor drug. Transgenic plant, as a bioreactor, has several advantages in which they are safe, inexpensive and can be administrated orally. Because the apc gene is from prokaryotic cyanobacteria, we at last chose the chloroplast of Chlamydomonas reinhardtii to product the using protein.In the first part of the thesis, we exploit the method of transforming the foreign gene into the chloroplast of Chlamydomonas reinhardtii, such as the parameter of biolistic deliver system, the method selecting and detecting transformations. At last we establish the Chlamydomonas reinhardtii chloroplast expression system in our lab.In the second part of the thesis, we construct a C. reinhardtii chloroplast expression vector: papc-S, which containing the apc cassette and selective marker-aadA cassette. The vector was introduced into the C. reinhardtii cell by biolistic method. After three round of selecting on spectinomycin plates, fifteen transformations were obtained. To determine the foreign gene had integrated into the genome of the C. reinhardtii chloroplast, two transformations were analysis by PCR and Southern-blotting. Because the primer was complementary to chloroplast 5'downstream sequence of chlL and 3' upstream sequence of apc respectively, the PCR and Southern-blotting result demonstrates that apc cassette have been integrated into the chloroplast genome of C. reinhardtii. In order to test the ape gene had expressed in the chloroplast of C. reinhardtii, the Western-blotting and ELISA wasdone. The result of Western and ELISA showed that the APC had expressed in the chloroplast of C. reinhardtii and accounted for 2-3% of the total soluble protein.Because the anti-tumor activity of recombination beta subunit of APC is better than the recombination of APC expressed in E. coli, in the third part of the thesis, we also construct the vector of C. reinhardtii chloroplast containing the beta subunit of APC gene. After the expression vector was introduced the cell of C. reinhardtii, the result of molecular testing show that the foreign gene had expressed in the cell and the level of foreign protein is about 3%.The two proteins have the activity of immunity, which indicate that the proper folding of the expressed proteins. Now, we is cooperating with China Pharmaceutical University to test the activity of the two recombination proteins expressed in the chloroplast of C. reinhardtii.