Sequence Analysis Of Endogenous Plasmid Of Lactobacillus Plantarum And Construction Of Its Expression Vector

Lactic acid bacteria can be colonized in animals and have outstanding physiological functions,improve the normal flora of the gastrointestinal tract,increase the conversion rate of livestock and poultry feed,and then increase the ratio of meat to material.China is a big consumer of meat food.Adding lactic acid bacteria preparations to the daily feed of livestock and poultry is of great significance to improving the quality of livestock and poultry meat,reducing the use of antibiotics and protecting people’s health.Plasmid stability plays an important role in microbial genetic engineering research and production.Only by constructing stable LAB vectors can the LAB vaccine be marketed.At present,many Lactobacillus plasmids have been sequenced and used to construct plasmid vectors.Lactobacillus plasmid replication is by rolling-circle(RC)and theta(θ)replication,and usually smaller plasmids(<12 kb)are replicated by rolling circle.Through the sequencing and analysis of the lactic acid bacteria plasmid,the replicon and stable gene of the lactic acid bacteria plasmid are the key to construct the LAB stable plasmid vector.The research on the isolation and functional analysis of natural plasmids of lactic acid bacteria has been carried out in China,and the improvement of Lactobacillus lactis by genetic engineering has been severely restricted.In this study,a plasmid was isolated from the excellent Lactobacillus plantarum,and the Escherichia coli-lactic acid bacteria shuttle plasmid was constructed based on the structure of the plasmid,and then a stable lactic acid bacteria expression vector was constructed,which laid a foundation for the oral vaccine research of lactic acid bacteria.The main contents and results of this study are as follows:1.In this study,the conventional lactic acid bacteria separation method:sample dilution,37℃bacterial liquid preliminary culture;strain streak separation;strain enrichment culture,microscopic examination;physiological and biochemical tests;bacterial 16Sr DNA identification,etc.,isolated a plant from sauerkraut Lactobacillus LP3,and has strong antibacterial ability to pathogenic bacteria.2.In this study,the complete sequence and functional analysis of the endogenous plasmid p LP3 in Lactobacillus plantarum LP3 isolated from sauerkraut was carried out.An Escherichia coli/Lactobacillus shuttle vector was constructed based on the p LP3,the chloramphenicol resistance gene from p SCPSP as a selection marker and the replicon of p UC19.At the same time,the host range,transformation rate and stability of the shuttle plasmid were studied.The result showed of the size of p LP3 was 2017bp and GC content was 37.48%.Based on the backbone of p LP3,we constructed an Escherichia coli/Lactic acid bacteria shuttle vector D-p LP3 and electroporated it into several strains of Lactic acid bacteria.The transformation efficiencies were ranged from 0.3×10~2～1.0×10~3cfu/μg.After 60 passages of serial passage,the shuttle plasmid has a loss rate of 30%in Lactobacillus plantarum with high genetic stability characteristics.The lactic acid bacteria expression vector D-p LP3-Pslp A-e GFP was further constructed by adding the promoter Pslp A and signal peptide of the Lactobacillus acidophilus and the e GFP target protein to the shuttle plasmid D-p LP3,and transformed into Lactobacillus plantarum and successfully expressed Green fluorescence of e GFP.3.The Cap protein of PCV2 has good immunogenicity and can stimulate the body to produce specific antibodies against PCV2.If the normal colonized Lactobacillus plantarum in the pig intestine is used as the expression host to express the Cap protein of PCV2,and apply it to the clinic.It may be of great significance for the prevention and control of circovirus infection.In order to detect the ability of the Lactobacillus acidophilus Pslp A promoter to express heterologous proteins in Lactobacillus plantarum,and to solve the disease caused by PCV2 infection in pigs in the breeding industry,this study utilized recombinant Lactobacillus plantarum D-p LP3-Pslp A-Cap It is made into an oral vaccine,which opens up a new way for genetic engineering to produce safe and convenient biological products.The ability of the expression vector to express a heterologous protein was detected by a(PCV2-CAP)ELISA specific detection kit.Experimental results:The lactic acid bacteria expression vector D-p LP3-Pslp A-e GFP was transformed into Lactobacillus plantarum,and the expression of fluorescent protein was successfully obtained.The PCV2-CAP protein ELISA assay showed an expression level of 30 ng/m L,and the D-p LP3-Pslp A vector had secretory expression ability.