Aspergillus Fumigatus Chitosanase Gene Cloning And Pichia Pastoris Expression

To make the chitosanase product industrialization.the study aimed to construct an effective and steady engineering bacteria.By comparing the classical total RNA extracting method.we get a improved RNA extracting method and use it we obtain the high quality total RNA.According to the N amino acid sequence of the Aspergillusfumigatus chitosanase and referring to the chitosanase gene sequence declared in the genebank,We design a pair primes.We use total RNA as template and obtain the aimed gene.we cut the aimed and the vacant vector pPIC9K with restriction endo—nuclease Not I and Avr II.then link them by ligase to produce the recombined vector and transform them into the DH5 a.Then choose the positive clone and send it to sequence.We linearize the recombined vector with the restriction endo—nuclease sal I.Then the recombined vector iS electro.transformed into the GS115.we choose and filter the positive strain by MD and strain plot PCR,induce the positive strain by methanol,check up the fermented broth with the SDS.PAGE electrophoresis and the chitosanase enzyme activity.This study through constructing expression vector and engineering bacteria make the chitosanase excretory express in the Pichia Pastoris.we carry out some study on bioactivities of chitosanase,the study make the foundation for the next step.