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...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。展开更多
目的探讨超敏C反应蛋白(hs-CRP)水平及rs1130864基因多态性与脑梗死(CI)的相关性。方法采用病例对照研究,对105例脑梗死患者和121例健康对照者进行研究。采用免疫透射比浊法测定hs-CRP,用多聚酶链式反应(PCR)和限制性片段长度多态性测定...目的探讨超敏C反应蛋白(hs-CRP)水平及rs1130864基因多态性与脑梗死(CI)的相关性。方法采用病例对照研究,对105例脑梗死患者和121例健康对照者进行研究。采用免疫透射比浊法测定hs-CRP,用多聚酶链式反应(PCR)和限制性片段长度多态性测定rs1130864基因多态性。患者入院后立即用美国国立卫生院神经功能缺损评分(National Institutes of Health Stroke Scale,NIHSS)对其进行神经功能测评。结果脑梗死组与对照组rs1130864基因型频率分布统计学无显著性差异(P>0.05)。rs1130864与脑梗死病情无关联。脑梗死多因素回归方程的主要危险因素包括高血压史、糖尿病史、吸烟史、HDL-C,hs-CRP水平。结论 rs1130864(1444C>T)基因多态性与脑梗死无关联。展开更多
Objective To explore the relationship between heat-shock protein 70-2 gene +1267A/G polymorphism(HSP70-2 gene +1267A/G polymorphism) and serum level of high sensitivity C-reactive protein(Hs-CRP) in patients with coro...Objective To explore the relationship between heat-shock protein 70-2 gene +1267A/G polymorphism(HSP70-2 gene +1267A/G polymorphism) and serum level of high sensitivity C-reactive protein(Hs-CRP) in patients with coronary artery disease(CAD).Methods Serum Hs-CRP level and HSP70-2 gene +1267A/G polymorphism were detected in 185 patients with CAD.HSP70-2 gene +1267A/G polymorphism was screened by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Results The genotype distribution of the HSP70-2 gene +1267A/G polymorphism was as follows: AA was in 24 of 185 patients(12.97%),AG in 93 of 185 patients(50.27%) and GG in 68 of 185 patients(36.76%).Patients with GG、AG subtype had higher Hs-CRP levels compared with carriers of the AA subtype(4.5±1.8mg/L,3.2±1.2mg/L and 2.7±1.1mg/L,respectively,P<0.05).Among patients who had Hs-CRP >3mg/L,GG,and AG gene subtypes had higher percentage than A allele homozygotes(92.6%,74.2% and 45.8%,respectively.χ2=17.75,28.04,P<0.05).Conclusions This study indicated G allele of HSP70-2 gene +1267A/G polymorphism may be an independent risk factor in systemic inflammation with stable CAD.展开更多
文摘目的探讨超敏C反应蛋白(hs-CRP)水平及rs1130864基因多态性与脑梗死(CI)的相关性。方法采用病例对照研究,对105例脑梗死患者和121例健康对照者进行研究。采用免疫透射比浊法测定hs-CRP,用多聚酶链式反应(PCR)和限制性片段长度多态性测定rs1130864基因多态性。患者入院后立即用美国国立卫生院神经功能缺损评分(National Institutes of Health Stroke Scale,NIHSS)对其进行神经功能测评。结果脑梗死组与对照组rs1130864基因型频率分布统计学无显著性差异(P>0.05)。rs1130864与脑梗死病情无关联。脑梗死多因素回归方程的主要危险因素包括高血压史、糖尿病史、吸烟史、HDL-C,hs-CRP水平。结论 rs1130864(1444C>T)基因多态性与脑梗死无关联。
文摘Objective To explore the relationship between heat-shock protein 70-2 gene +1267A/G polymorphism(HSP70-2 gene +1267A/G polymorphism) and serum level of high sensitivity C-reactive protein(Hs-CRP) in patients with coronary artery disease(CAD).Methods Serum Hs-CRP level and HSP70-2 gene +1267A/G polymorphism were detected in 185 patients with CAD.HSP70-2 gene +1267A/G polymorphism was screened by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Results The genotype distribution of the HSP70-2 gene +1267A/G polymorphism was as follows: AA was in 24 of 185 patients(12.97%),AG in 93 of 185 patients(50.27%) and GG in 68 of 185 patients(36.76%).Patients with GG、AG subtype had higher Hs-CRP levels compared with carriers of the AA subtype(4.5±1.8mg/L,3.2±1.2mg/L and 2.7±1.1mg/L,respectively,P<0.05).Among patients who had Hs-CRP >3mg/L,GG,and AG gene subtypes had higher percentage than A allele homozygotes(92.6%,74.2% and 45.8%,respectively.χ2=17.75,28.04,P<0.05).Conclusions This study indicated G allele of HSP70-2 gene +1267A/G polymorphism may be an independent risk factor in systemic inflammation with stable CAD.
