Construction Of The Fusion Expression Vector And The Clone And Expression Of α-amylase Gene For Lactic Acid Bacteria

As important industrial microorganisms, Lactic acid bacteria has been widely used in manyindustries such as animal husbandry, food, pharmaceutical and so on. At present, some of them wereregarded as typical probiotics. With the deepening study on physiological and biochemical of lacticacid bacteria, researchers intend to reveal the activity mechanism of colonization, distribution,survival and probiotic property of lactic acid bacteria desperately. Therefor, it is necessary that usingmolecular biology technology to construct the recombinant strain by which could play an role asindicator. The gene dsred2encodes a kind of red fluorescent proteingene which could be detected bynaked eyes after maturity. The bright red of strains was obvious in the sunlight. Tracking the Lacticacid bacteria marked with dsred2, that could be helpful to clarify the physiological mechanism oflactic acid bacteria and the role of the functional gene．In this study, based on a shuttle vector of E．coli and lactic acid bacteria pMG36e, a fussionexpression vector marked by dsred2was constructed and it was named pMG36e-dsred2. Thenα-amylase gene amy was subcoloned on as pMG36e-dsred2a report gene and we got a recombinantstrain expressing red fluorescence protein and α-amylase at the same time.According to the sequence of dsred2published on Genebank,a pair of primer were designedand synthesized.Then wo plused two restriction sites of Xba I and HindIII at the upstream anddownstream of the primer.The whole coding sequence of dsred2gene of the plasmid pPIC9k-dsred2was amplified and then connected it to the vector pMD19-T. Use XbaI and Hind III to digest thevector pMG36e and pMD19-T at the same time.Furify and connect the linear pMG36e and the smallfragments dsred2with T4ligase,so we get the recombinant vector pMG36e-dsred2then transforminto Escherichia coli DH5α.The coding sequence of α-amylase gene which has not the stop codon was amplified by PCRbased on the Bacillus licheniformis genome.As we has plused the two restriction sites of SacⅠandXba Ⅰ at the upstream and downstream of the gene, digest the vector pMG36e-dsred2andpEASY-T1-amy with the two restriction enzyme. Furify and connect the linear pMG36e-dsred2andthe small fragments amy with T4ligase,then we get the fusion expression vector pMG36e-dsred2-amy.Transforme the new vector into Lactobacillus casei by electroporation.The study found that the gene dsred2and fusion gene dsred2-amy gene all can be expressed inEscherichia coli and LAB．The expression of red fluorescent protein in E.coli (pMD19T-dsred2) morethan E.coli (pMG36e-dsred2). No matter The fusion protein was expressed in E.coli or LAB, it showed the activity of dsred2and amy. And there is no significant differnce of the expression betweenfused and individual. SDS-PAGE of fusion protein confirmed that dsred2gene was fused withamygene successfully, and the molecular weight of dsred2and fusion protein dsred2-amy were24.8kDaand81.5kDa, respectively.In this study，we constructed the vector pMG36e-dsred2which can express the fusion protein inL.acid, successfully, and detected the fluorescence activity of expression in the recombine strainsE.coli (dsred2) and LAB acid (dsred2)．Furthermore, we used the a-amylase gene to testify theexpression vector of the fusion protein. Our research supplied a new method to analyze thecolonization, distribution and survive of lactia acid bacteria in vivo. In addition, the researchachievement lay the foundation of revealing the secretory expression condition (in the cell，on the cellwall or the out of the cell) of lactia acid bacteria and detecting the activity of heterologous protein.