The Study Of Biological Characteristic Of A Lactobacillus Casei Phage L J And The Identification Of Promoter And Replicon

Lactic acid bacteria(LAB,lactic acid bacteria)is a gram positive facultative anaerobic bacteria,including Lactococcus lactis,Lactobacillus,Bifidobacterium and other ten generas.It has long been recognized as a security level(GRAS,generally recognized as safe)of the microorganisms in the food industry for a long-term application domain of each collar.With the development and application of molecular biology in the field of lactic acid bacteria,lactic acid bacteria expressing exogenous protective antigen,which has been widely used.Many studies have shown that recombinant lactic acid bacteria have good application in food and pharmaceutical industry.Useing double-layer agar method with Lactobacillus casei as host bacteria,we isolate a Lactobacillus casei bacteriophage LJ from the Northeast sauerkraut.Phage LJ composite with apolyhedral head and a non contractile soft flexible tail,which head diameter was about 37.9nm,and the tail length was about 216.83nm.According to morphology classification,its return to the Siphoviridae family B1.The phage genome does not contain sticky ends,the packaging mechanism is packaging,named pac-type.One step growth curve of phage LJ were 165min,rolysis time 105min,adsorption amount of bacteriophage was 121PFU/cell.The phage was dependent on Ca2 and Mg2,especially Ca2+ was better than Mg2 with good stability to phage cracking.The results showed that the phage is sensitive to temperature,and temperature affect the adsorption activity.High temperature is not suitable for the phage adsorption,and the phage would be inactivative.When the phage was exposed to UV light conditions,the phage completely lost activity within 20min.Phage LJ has a certain extent acid and alkali tolerance,maintain a certain activity within pH3-12 range.The phage LJ has little of tolerance with ethanol and isopropanol.The phage LJ will be inactivated under 75%ethanol and isopropanol in 30min.The phage LJ will be inactivated under 70? in 30min.Ca2+ and/or Mg2+ would promote the adsorption with the host.In this study,the total phage is divided into eight section for amplification of the phage LJ genome with eight pairs of primers,each gene is about 5000bp.The PCR products were purified for the sequencing.Finally,splicing sequence of the bacteriophage genome was about 44 348bps,which GC content is 44.78%.Phage contains 65 open reading frames,including 57 with function annotation after prediction.Through the analysis of gene purification tree,phage LJ,Lactobacillus rhamnosus phage Lrml and Lactobacillus casei A2 phage genome have the highest similarity of phage LJ genome according to the function of genes arrangement together.The phage head,tail,copy,cleavage and packaging module was similar with Lactobacillus phage and Lactobacillus Lrml.Lactobacillus casei phage A2 modules have both with the composition arrangement order and alignment similarity analysis,whcih head,tail,replication and lysis modules module of the phage LJ has high degree of similarity with other two strains phage.The experiment was designed to obtain phage structure gene promoter with 8 mRNAs of 5' end sequences.The transcription initiation site of phage structural genes was used to analysis the promoter sequence.At the same time the software predict the phage promoter sequence of the genome,total promoter sequences were 8 promoters.The RACE method and software prediction method promoters were constructed into the vector pPT7g10-p plasmid,which p was with an anchoring protein.16 promoter probe vector were electroporated into Lactobacillus casei,verified promoter activity by the Westernblot.Westernblot confirmed the promoter activity with anchor-pgsa polyclonal antibody.There were 7 promoter sequences with moderate to strong promoter activity.The expression of anchor-pgsa was HK1,HK3,TTM,PPP,pFlpab,35800and Sigma promoter.The pFlpab,35800 and sigma promoter were found by software prediction.The structural gene promoter HK1 and HK3,TTM and PPP were found by 5' rapid amplification of cDNA ends(RACE).The structural proteins respectively for phage tail binding protein(HK1),phage capsid protein(HK3),phage major tail protein(TTM)and phage particle protein(PPP).Experiments show that phage structural gene promoters have a higher promoter activity,the three structural genes of medium strong promoter.Promoter prediction software confirmed eight predicted promoter with in three promoter sequences with promoter activity,and promoter activity were higher than part of the structure gene promoters activity.In addition,using the identified weak promoter ET and strong promoter Sigma,recombinant Lactobacillus expression system using EGFP as model antigen was respectively constructed to further verify the promoter activity of ET and Sigma,followed by the idenfication of flow cytometry,Western blot and indirect immunofluorescence.The results showed that the promoter activity of the promoter Sigma was significantly stronger than that of ET,which was consistent with the previous identification results.Phage replication module including single strand binding protein gene(SSB),phage helicase(DnaB and DnaC)were amplified by PCR.The SSB was named replication genes constructed into the Lactobacillus expression vector of pPG612 plasmid.pPG612 plasmid was with RepA and RepC of two replication protein at the same time.The replication of phage was related with the genes of SSB.The chloramphenicol resistance gene on the plasmid was the targeted gene with the real-time PCR,using Lactobacillus genome 16sRNA gene as a reference gene.The Lactobacillus casei containing plasmid pPG612 was the control group.The experimental results show that the replication gene increased copy number of plasimd.The highest copy number of plasimd was after 14h culture,which copy number of plasmid is about 14.8.The copy number of plasmid began to decrease at 14 h,and reach to the minimum at 22h.The study found seven moderate intensity of lactobacilli plasmid promoter sequence.Replication gene of phage would improve the copy number in the host of Lactobacillus casei.The research of lactic acid bacteria phage derived promoter and replicon,which promote the genetic engineering research,and laid the good foundation.