Cloning And Expression Of Peptidoglycan Recognition Protein And Lysozyme Gene In Cow And Correlation Analysis Between SNP And SCS

Dairy cows mastitis is inflammation in Mammary gland which mainly caused pathogens,and was one of the most damaging diseases in dairy production. Among the pathogenscausing mastitis, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae andStreptococcus dysgalactiae were most common pathogenic bacteria. In natural immunesystem of cow, peptidoglycan recognition protein and lysozyme are two kinds of veryimportant pattern recognition receptors (PRR), which can recognize the peptidoglycan (a kindof Pathogen Associated Molecular Pattern, PAMP) derived from pathogens, and playimportant roles in defensing pathogen invasion and eliminating pathogens.In this research, the genomic DNA of cow was used as template for amplifying genes ofpeptidoglycan recognition protein. By sequencing the PCR product of peptidoglycanrecognition protein, the polymorphic site in DNA sequence was identified. Then genotype ofpolymorphic site in each individual was identified with the PCR-RFLP method. Byperforming the correlation analysis between SNP and SCS, milk production traits, the SNPaffecting mastitis resistance and milk production traits in cow can be screened, which canprovide theoretical basis for disease resistance breeding in cow. On the other hand, with thehelp of bioinformatics, molecular biology and genetic engineering techniques, all four genesof peptidoglycan recognition protein family and5genes of subtypes in lysozyme family wascloned and expressed in Yeast eukaryotic expression system and/or E. coli prokaryoticexpression system. To identify the antimicrobial activity of soluble recombinant targetproteins, the antibacterial test against5species of common pathogens in mastitis wasperformed, which can provide theoretical and technical basis for research and development ofnew type of antimicrobial agents used in prevention or treatment of mastitis. The main resultswere showed as following:1. Polymorphism in PGLYRP1gene and correlation analysis between SNP andSCS, milk production traits A total of ten single nucleotide polymorphism (SNP) loci were identified in PGLYRP1gene. The association analysis of single SNP locus showed that T-35A, T-12G and G+102Cwere significantly associated (P<0.05) with somatic cell score (SCS), while G+102C andG+649C were significantly associated (P<0.05) with average daily milk yield. Associationanalysis between combined haplotypes and SCS, milk production traits indicated that H3H3was associated with the lower SCS (P<0.01), and H2H2was associated with the lowerAverage daily milk yield (P<0.01). It is concluded that haplotype H3H3could be used asgenetic marker in molecular marker-assisted selection2. Polymorphism in PGLYRP2gene and correlation analysis between SNP andSCS, milk production traitsA total of five SNP loci were identified in PGLYRP2gene. The association analysis ofsingle SNP locus showed that the C+4867T locus was significantly associated (P<0.05) withsomatic cell score(SCS). Surprisingly, all loci were significantly associated (P<0.01orP<0.05) with fat rate. Association analysis between combined genotypes and SCS and milkproduction traits indicated that H2H2was associated with higher fat rate (P<0.05). It isconcluded that SNP in PGLYRP2gene was associated with SCS and fat rate, and furtherresearch is needed to identify mechanism in which PGLYRP2influenced fat rate in milk3. Polymorphism in PGLYRP3and PGLYRP4gene and correlation analysisbetween SNP and SCS, milk production traitsA total of three SNP loci were identified in coding region of PGLYRP3gene and twoSNP loci were identified in coding region of PGLYRP4gene. The association analysis ofsingle SNP locus showed that the only P3-T870C locus was significantly associated (P<0.05)with Average daily milk yield in PGLYRP3gene. And in PGLYRP4gene, P4-A536G wassignificantly (P<0.01or P<0.05) associated with SCS, fat rate and protein rate. All thecombined haplotypes of PGLYRP3were not associated with any traits. Combined haplotypesof PGLYRP4were associated with SCS and fat rate. Incorporating combined haplotypes ofPGLYRP3and PGLYRP4were not associated with any traits. It is concluded that P4-A536Gin PGLYRP4gene was important SNP locus, with potential to be used in molecularmarker-assisted breeding.4. Cloning, sequence analysis, and eukaryotic expression of PGLYRP1gene anddetection of antibacterial activity of recombinant PGLYRP1cDNA from cell in blood was used as template for amplifying PGLYRP1gene.Linearized pPIC9K-PGLYRP1was recombined with genomic DNA of GS115with method of electric shock. After screening with4mg/mL G418, the recombinant yeast strains withmulti-copy of PGLYRP1were obtained and soluble recombinant PGLYRP1were identifiedby Tricine-SDS-PAGE. By concentrating with ammonium sulfate in60%saturation andpurifying with Ni-NTA, recombinant PGLYRP1was obtained at concentration of0.3mg/mL.In antibacterial test for exploring the antimicrobial activity of recombinant PGLYRP1againstcommon pathogens in mastitis, PGLYRP1had better antibacterial activity on Staphylococcusaureus and Streptococcus agalactiae than Streptococcus dysgalactiae, and had no antibacterialactivity on Escherichia coli and Salmonella typhimurium.5. Cloning, sequence analysis, and eukaryotic expression of PGLYRP2, PGLYRP3S,PGLYRP3L and PGLYRP4gene.PGLYRP2was amplified with cDNA template from liver and PGLYRP3as well asPGLYRP4was amplified with cDNA template from esophagus. Two kinds of alternativesplicing of PGLYRP3was identified, and named them as PGLYRP3S(for short) andPGLYRP3L(for long), the digested fragment of PGLYRP2, PGLYRP3S, PGLYRP3L andPGLYRP4were ligated with pPIC9K, pGAPZαA and pPICZαA respectively. Linearizedrecombinant expression vectors with pPIC9K were recombined with genomic DNA of GS115and KM71respectively. At the same times, linearized recombinant expression vectors withpGAPZαA and pPICZαA were recombined with genomic DNA of X33respectively.Detection of recombinant protein with SDS-PAGE indicated that PGLYRP2, PGLYRP3S,PGLYRP3L and PGLYRP4did not express in any recombinant yeast stains. It is concludedthat PGLYRP2, PGLYRP3S, PGLYRP3L and PGLYRP4may not be appropriately expressedin pichia pastoris expression system.6. Prokaryotic expression of PGLYRP2, PGLYRP3S, PGLYRP3L and PGLYRP4gene.The digested fragment of PGLYRP2, PGLYRP3S, PGLYRP3L and PGLYRP4wereligated with Pet22b, Pet31b and pet43.1b respectively. On the other hand, the digestedfragment of PGLYRP2was ligated with Pet32a. All the recombinant expression vectors weretransformed to Rosetta-gami B(DE3)plysS. After inductive cultivation at22℃, Detection ofrecombinant protein with SDS-PAGE indicated that PGLYRP2, PGLYRP3S, PGLYRP3L andPGLYRP4expressed in Pet22b, Pet31b were all insoluble. PGLYRP2in Pet32a was soluble inlow percentage. PGLYRP2, PGLYRP3S, PGLYRP3L and PGLYRP4expressed in Pet43.1bwere soluble in relatively high percentage. But all the recombinant proteins expressed inPet43.1b were not purified successfully. The reason may be Nus tag was too big, two His tagsin recombinant protein were folded into interior of protein. 7. Cloning and eukaryotic expression of6subtypes of lysozyme gene, and detectionof antibacterial activity of recombinant lysozymesSignal peptide prediction of6subtypes of lysozyme indicated that all subtype oflysozymes had same length of signal peptide. By sequencing the DNA sequence of6subtypeof lysozyme, LYZ-Milk was not amplified, maybe caused by the low expression in mammarygland compared to LYZ14D. The other5subtypes (LYZ3, LYZ2, LYZ14D, LYSB and LYZ) oflysozyme were cloned successfully and ligated with pPIC9K after digesting. Aftertransforming into GS115, screening with4mg/mL G418, Inductive cultivation andTricine-SDS-PAGE detection,5subtypes of lysozymes were all soluble. But the express levelof LYSB was very low. In the purification of recombinant lysozyme, only four subtypes(LYZ3, LYZ2, LYZ14D and LYZ) of lysozymes were purified successfully. In antibacterialtest for exploring the antimicrobial activity of recombinant PGLYRP1against commonpathogens in mastitis, Lysozymes from stomach (LYZ3and LYZ2) had better antibacterialeffect than which from other tissues (LYZ14D and LYZ). At same concentration of0.25mg/mL, LYZ3and LYZ2had better antibacterial effect on Staphylococcus aureus thanEscherichia coli, and had no antibacterial effect on Streptococcus agalactiae, Streptococcusdysgalactiae and Salmonella typhimurium. LYZ14D and LYZ had no antibacterial effect onall5pathogens. It is concluded that LYZ3and LYZ2derived from stomach had potential as adrμg for the treatment of mastitis.