Cloning The Gene Of Chymopapain And Transformation Into Lactobacillus Acidophilus

Rennin is an important enzyme preparation used in the cheese manufacturing industry extensively. Its production value possesses 15% of the enzyme preparation of the whole world. As the production of the cheese increases, the quantity of chymosin we want is also increasing sharply, so the bovine chymosin can not meet the need. At the same time, the animal infection always reakes out, therefore the consumer is afraid of the chymosin deriving from animal. In addition, the vegetarian think that the cheese that made with bovine chymosin is unacceptable. So many countries are all seeking a substitute for bovine chymosin. The chymopapain is a kind of plant chymosin that has the curd function. Clone the gene of chymopapain and transform into Lactobacillus acidophilus ATCC4356 that is probiotic. Be used for the cheese production. It is of great significance for solving the lack of chymosin and satisfying the demand of the special crowd. At the same time, it is benefit for health, and layes a foundation for producing the commercial protein.The research extracted the total RNA from green Carica papaya L. with Trizol reagent kit, and synthesized the first chain of cDNA with reverse transcription. Design specificity primer carring recognition site of restriction enzyme SacI and XbaI, optimize condition of PCR, amplify and obtain the gene of chymopapain which is ligated into pMD18-T simple vector. Then we obtained the recombined plasmid pMD18-T+. The plasmid pMD18-T+ was transformed into E.coli. JM 109 and sequenced. Comparing the sequencing result with the nucleic acid sequence of the chymopapain pubilicized on GenBank (AJ131996), more than 98% of sequence is identical. Digest the plasmid pMD18-T+ and the plasmid pMG36e respectively with restriction enzyme SacI and XbaI, and recover the purpose gene and the vector segment. Then ligate the two segments, and obtain the recombine plasmid pMG36e+. Activate the receptor strains Lactobacillus acidophilus ATCC4356, and optimize the transformation parameters for electrotransformation. Transform Lactobacillus acidophilus ATCC4356 and obtain 6 transformants. Identify the result with the methods of PCR and being digested by restriction enzymes SacI and XbaI. The recombined plasmid pMG36e+ was transformed into Lactobacillus acidophilus ATCC4356 successfully.