Study On Heredity Stabilitary Of Ac-hIL12 And Expression Of HIL-12 In Pichia Pastoris

Baculovirus expression vector system (BEVS) is a most valuable system of expressing foreign protein. However, a major drawback in the lage-scale production of baculoviruses for heterologous protein production is so-called passage effect. This effect is notable as a significant drop in production after prolonged virus passaging in insect cell culture that results from the accumulation of defective interfering particle (DIP).In this research,the heredity stability of a recombinant baculovirus expressing human interleukin-12 was studied.The strain purified from a baculovirus expressing recombinant human interleukin-12 by plaque selection was serially and undilutedly passaged up to 55th generation in Spodoptera frugiperda 9(SF9) cells. Intracellular viral (ICV) DNAs were extracted from the infected SF9 cells of P₁5, P₂5, P₃5, P₄5, P₅5 generation of recombinant baculovirus.A 2.0kb DNA fragment including sequences of P35 cDNA, polyhedrin promoter, P10 promoter and P40 cDNA was amplified by PCR.The sequence analysis indicates that there is no mutation in 2.0kb nucleotide sequence during the P15 to P35 passages.However,the point mutation was detected at three nucleotide residues sites in P35 cDNA sequence (461T→C, 517A→G and 630C→T) and one nucleotide "T" was inserted between +1 position of polyhedrin promoter and the upstream of BamHI recoganizing site at P₄5 generation. Passaging to P55, the insertional mutation (-136T-135),deleton mutation (-121T) and point mutation (-168G→T) happened in the P10 promoter cassette besides the above mutations.These results show that serial and undiluted passaging of engineering baculovirus can result in foreign genemutation.Abstract2: Today Pichia expression system has been one of the most attactive systems for expressing foreign protein.A variety of heterologous proteins have been produced in this highly successful system.In the present study,the p40 cDNA was amplified from pCR40 by PCR and cloned to pPIC6aC.The recombinant plasmid pPIC6aC/p40 was constructed.The p40 cDNA including a-factor signal sequence,so called p40a,was amplified from pPIC6aC/p40 by PCR.Meanwhile,p35 cDNA and IRES sequences were amplified from plasmids pCR35 and pIRES2-EGFP by PCR respectively. The p35cDNA( Clal & S/?d),IRES (Spel & Ncol) and p40a (Ncol & NotI) fragments were cloned to pPIC6aC(C/aI & NotT) together. The plasmid pPIC6aC/p35-IRES-p40a was successfully constructed and transformed to Pichia pastoris X-33.10 large clones were picked from labeled YPDS or YPD containing 300ug/ml blasticidin plate and purified on fresh YPD plates containing the appropriate concentration of blasticidin.After the recombinant yeasts had been induced for 72h with 0.5% methanol,the medium were detected using ELISA kit.The results indicate the foreign genes,p35cDNA and p40cDNA,were successfully expressed, and the EMCV IRES can perform its cap-independent translating function in Pichia pastoris. The production is 30ng/L.The expression of the recombinant yeast was studied in different time and in different conditions.