Establishment Of A Cross-priming Amplification Assay Of Lawsonia Intracellularis And Analysis And Prokaryotic Expression Of LI0710 Gene

Lawsonia intracellularis(LI),as a pathogenic bacterium for Porcine proliferative enteropathy(PPE),has a simple way of transmission,high infection rate,easy to cause secondary infection and low mortality,which will cause great economic loss to the farmers.With the advent of prohibiting the addition of antibiotics in feed in China,early detection and prevention and control is an effective way to control the large-scale spread of Lawsonia intracellularis.The purpose of this study is to establish a method for the rapid and accurate detection of Lawsonia intracellularis infection.The prokaryotic expression of intracellular Lawsenia LI0710 protein laid a theoretical foundation for the development of genetic engineering vaccine and the establishment of serological diagnosis methods in the future.1 Establishment and preliminary application of Cross-priming amplification of Lawsonia intracellularis.According to Lawsonia intracellularis genes(Gen Bank:CP004029.1),specific primers for PCR and CPA were designed respectively.The reaction conditions were optimized by adjusting annealing temperature,reaction temperature,reaction time,cross primers,peeling primer concentration ratio,d NTPs concentration,magnesium ion concentration and betaine concentration,and finally the reaction system was determined.Then,sensitivity,specificity,reproducibility tests and clinical sample tests were used for validation.According to the sensitivity test,the minimum copy number of the samples detected by PCR method was3.69×10~3copies,and that by CPA method was 3.69×10~1copies.Specific tests showed that PCR and CPA were negative for Escherichia coli,Brachyspira hyodysenteriae,Streptococcus suis,Staphylococcus aureus and Salmonella.Visual inspection of the product of the CPA,according to the results of positive product appear obvious turbidity,in natural light,under ultraviolet light,join SYBR GreenⅠnucleic acid masculine product can emit Green fluorescent dye,negative product no fluorescence.The PCR and CPA methods were used to detect 267 samples collected from Guangdong Province.The results showed that the positive rate of PCR detection was 27.3%(73/267),and the CAP was 41.9%(112/267).A total of 73 samples were tested positive by both methods,while 155 samples were negative.In addition,39samples were tested positive by CPA and negative by PCR.Several positive samples detected by both methods were selected for cloning transformation and sequencing,and the sequencing results showed they were Lawsonia intracellularis.Some of the samples that were positive by CPA but negative by PCR were selected for nested PCR and cloned and sequenced after completion of the test.2 Analysis and prokaryotic expression of LI0710 geneAccording to the complete gene sequence of A uploaded on NCBI,the LI0710 gene sequence was selected,and the protein expressed by LI0710 was predicted and analyzed by using molecular biology software.Based on the results of the analysis and the principles of codon optimization,a nucleotide sequence in LI0710 gene was synthesized,and Bam HI and Xhol restriction sites were added into the nucleotide sequence.Sequencing analysis of synthetic sequence,build p ET28a-LI0710 recombinant plasmid,and then to transform recombinant plasmid into BL21(DE3)cells,by inducing purification,successfully expressed proteins,using SDS-PAGE,after electrophoresis,the positive serum as a resistance to Western Blotting detection,the size of the test results show that the protein is about 20 kda,and forecast the result of match.