Gene Induction And Expression System Of Streptococcus Mutans And Its Application

Gene expression system plays an important role in metabolic pathway,signal pathway and gene expression regulation.It is widely used in biology,food,industry and medicine.Prokaryotic cells and eukaryotic cells are used as the expression system to express the target gene sequence to produce protein vaccine,nucleic acid vaccine and biological enzyme preparation.Induced gene expression system is a specific,efficient and dose-dependent system,which has been successfully applied in the production of enzymes,polypeptides,intermediate metabolites and other expression products,and has greatly promoted the development of food,medicine,health care and industry.By using strong promoters to integrate cloned genes into chromosomes,efficient expression vectors were constructed to achieve efficient expression of foreign proteins through different induction methods.At present,there have been relevant studies on the construction of the induction system of Streptococcus mutans,but the combination of this system and transposition system to explore the functions of some related genes is still insufficient.In this paper,?Streptococcus mutans?UA159 was taken as the research object to construct an inducible expression system that can effectively control gene expression and be combined with transposition system for this bacterium.The main research results are as follows:?1?In the heart of the S.mutans UA159 transposable element with the existing two xylose S2 and S1 induced system were constructed two translocation carrier pLH475?pLH145::gyrAp::xylR::xylAp::xylA?::marc9::ermAM?and pLH630?pLH145::gyrAp::xy/R::ldhp::xylA?::marc9::ermAM?,induced by xylose experiment and found that both systems can not meet without inducer and low expression level,high expression at induction.The former had no transposition after the addition of xylose,while the latter had a large number of transposers without the addition of xylose and the transposon point was single.?2?Construction of a new inducible expression system S3.Induced by xylose repressor gene modification after xylR RBS area,construct a new guidance system,then use this system to construct a plasmid pLH632 fluorescein reporter?pLH421::gyrAp::xylR::ldhp::xylA?::luc?,through the luciferase activity test results show that the system can lower levels in the absence of inducer expression,is lower than S1 expression level 4 times,after join xylose induced,a new guidance system expression level is higher than the amount of the expression of S2 is about 2.8 times higher,an induced expression system with low basic expression level and high induced expression level was successfully constructed.Induced expression system.?3?The new S3 and transposable elements combine to build translocation carrier pLH631?pLH145::gyrAp::xlR'::ldhp::xylA?::marc9::ermAM?into s.mutans UA159,xylose induction experiments results show that after around not join xylose induced with little or no transposons,when adding 0.4%xylose reached the maximum number-of transposons,translocation rate and maximum of 3%.The results of transposon sequencing after some serial processing showed that the transposon insertion sites had diversity.?4?The use of translocation system screening resistant mutant strains,the translocation carrier pLH631 into S.mutans in UA159 xylose translocation occurred after induction,will change the mutant strains of coating on the tablet containing acetic acid chloride is resistance screening mutant strains of acetic acid chloride is set,select the mutant strains of insert genes are SMU₁349,and after the trials showed that insertion mutation can be steady heredity.