cya/crp基因缺失对鸡白痢沙门菌生物学特性影响的研究

The effect of cya/crp deletion in Salmonella pullorum on its biological characteristics

为了解crp/cya基因对鸡白痢沙门菌毒力和生物学特性的影响,本研究构建缺失1182 bp cya基因的重组自杀性质粒pREΔcya,并经酶切和测序鉴定正确后,转化大肠杆菌χ7213作为供体菌,分别以鸡白痢沙门菌C79-13株和本研究室前期构建的其crp基因缺失株C79-13Δcrp(简写为Δcrp)为受体菌,利用重组自杀性质粒介导的同源重组技术构建C79-13菌株的单基因缺失株Δcya以及双基因缺失株ΔcrpΔcya,并均经PCR和测序鉴定。将这3株菌连续传60代后每隔10代分别利用PCR鉴定cya基因缺失的遗传稳定性;利用玻片凝集试验鉴定各菌株的血清型,并鉴定各菌株的生化反应特性;绘制各菌株的生长曲线分析缺失株与亲本株的生长特性;通过微量结晶紫法检测各菌株的生物被膜(BF)形成能力。PCR和测序鉴定结果显示Δcya、ΔcrpΔcya均正确构建,且cya基因缺失均具有稳定的遗传性。血清型及生化鉴定结果显示,菌株Δcya、Δcrp、ΔcrpΔcya均保留了C79-13的O9血清型,而失去了发酵蔗糖、鼠李糖、葡萄糖、麦芽糖等糖类的能力。生长曲线及BF形成能力的检测结果显示,各缺失株的生长速度较亲本株相比均显著降低,尤其双基因缺失株ΔcrpΔcya的生长速度极显著下降(P<0.01),且基因缺失株BF的形成能力均极显著降低(P<0.01),其中双基因缺失株BF的形成能力更低。将3株缺失菌及亲本菌分别以不同的剂量(5×10^(8)cfu/只~7×10^(11)cfu/只)经腹腔注射感染雏鸡进行毒力试验,计算各菌株对雏鸡的半数致死量(LD_(50))。将各缺失株以10^(7) cfu/mL两次经灌服免疫雏鸡,100μL/只,二免后7 d各组雏鸡均以5.0×10^(8)cfu/mL经腹腔注射亲本菌株C79-13进行攻毒试验,500μL/只,在42 d的观察期内观察并记录各组鸡的发病及死亡情况,计算各基因缺失株对雏鸡的免疫保护率。结果显示,缺失株Δcya、Δcrp、ΔcrpΔcya对雏鸡的LD_(50)分别为2.84×10^(9) cfu/mL、1.41×10^(10)cfu/mL、2.51×10^(11)cfu/mL,与亲本菌株C79-13相比,各缺失株LD_(50)分别升高约1.1×10^(3)倍、5.7×10^(3)倍、1.0×10^(5)倍;各组雏鸡攻毒后出现精神沉郁症状,且有部分鸡死亡,PBS对照组鸡出现排稀便症状,且全部死亡。Δcya、Δcrp及ΔcrpΔcya对雏鸡的免疫保护率分别为60%、80%、50%。表明各基因缺失株均能够对雏鸡提供一定的免疫保护效果,且单基因缺失株Δcrp的免疫效果最好。本研究首次对鸡白痢沙门菌crp、cya基因生物学功能做了初步研究,并证实缺失株Δcrp有望作为一种鸡白痢沙门菌基因缺失疫苗的候选菌株,为研制安全有效的沙门菌弱毒活疫苗奠定了基础。

To investigate the effects of crp and cya gene deletion on virulence and biological characteristics of Salmonella pullorum,the recombinant suicide plasmid pREΔcya containing a deletion of 1182bp cya gene was constructed and transformed into Escherichia coliχ7213 which acted as the donor bacteria.The parental strain of Salmonella pullorum C79-13 and the formerly constructed crp mutant deletion strain C79-13Δcrp were used as the recipient bacteria.The C79-13Δcya and C79-13ΔcyaΔcrp mutants were constructed using the recombinant suicide plasmid-mediated allele exchange technology and confirmed by PCR and sequencing.The genetic stability of cya gene deletion was identified by PCR every 10 generations after 60 generations of continuous passage of these three strains.The serotype of each strain was identified by slide agglutination test,and the biochemical reaction characteristics of each strain were identified.The growth curve of each strain was drawn to analyze the growth characteristics of the deletion strain and the parent strain;the biofilm(BF)formation ability of different strains was detected by microcrystal violet method.The results showed that the C79-13Δcya and C79-13ΔcrpΔcya mutants were successfully constructed and cya gene deletion has stable heredity.The C79-13Δcya,C79-13Δcrp and C79-13ΔcyaΔcrp mutants retained the O9 serotype of C79-13,while lost the ability to ferment sucrose,rhamnose,glucose,maltose and other sugars.The results of growth curve and BF formation ability test showed that the growth rate of each deletion strain was significantly lower than that of the parent strain,especially the growth rate of the double gene deletion strainΔcrpΔcya was significantly decreased(P<0.01),the BF formation ability of the gene deletion strains was significantly decreased(P<0.01),and the BF formation ability of the double gene deletion strain was lower.The chickens were intraperitoneally infected with the three deletion mutants and the parental strain at different doses(5×10^(8)cfu/chick-7×10^(11)cfu/chick)for virulence experiments,and the median lethal dose(LD_(50))of each strain to chickens was calculated.The chickens were immunized by gavage twice with 10^(7)cfu/mL of each deletion strain,100μL/chick,and 7 days after the second immunization,the chickens in each group were intraperitoneally challenged with 5.0×10^(8)cfu/mL of the parent strain C79-13,500μL/chick.The incidence and mortality of chickens in each group were observed and recorded during the observation period of 42 days,and the immune protection rate of each gene deletion mutant on chickens was calculated.The results showed that LD_(50)of C79-13Δcya,C79-13Δcrp and C79-13ΔcyaΔcrp mutants were 2.84×10^(9)cfu/mL,1.41×10^(10)cfu/mL and 2.51×10^(11)cfu/mL,respectively,and the LD_(50)were increased by 1.1×10^(3),5.7×10^(3),and 1.0×10^(5)times,respectively,compared with the parental strain C79-13.The results of the challenge tests showed that the chickens in each group showed symptoms of depression after challenge,and some chickens died.The chickens in the PBS control group showed symptoms of diarrhea,and all died.The immune protection rates of the three deletion mutants were 60%,80%and 50%,respectively,indicating that each gene deletion strain can provide certain immune protection effect on chickens,and the single gene deletion strainΔcrp has the best immune efficacy.In this study,the biological functions of crp and cya genes of Salmonella pullorum were studied for the first time,and it was confirmed that the deletion strainΔcrp was expected to be a candidate for gene deletion vaccine of Salmonella pullorum,which laid a foundation for the development of safe and effective live attenuated salmonella vaccine.