致奶牛乳腺炎金黄色葡萄球菌Csa1A、Hlɑ和EsxA-B重组蛋白的表达及免疫效果的评价

Expression and immune efficacy evaluation of recombinant protein Csa1A, Hlɑ and EsxA-B of Staphylococcus aureus from dairy cow mastitis

为评价金黄色葡萄球菌(S. aureus)Csa1A、Hlɑ和EsxA-B蛋白的免疫保护作用,本研究构建了重组质粒p GEX-4T-2-Csa1A、pET-22b-Hlα、pGEX-4T-2-EsxA-B并分别转化大肠杆菌BL21(DE3),经IPTG诱导并利用相应层析柱纯化后,采用SDS-PAGE检测重组蛋白的表达。结果显示,获得了纯度较高的重组蛋白Csa1A(rCsa1A)、Hlɑ(r Hlɑ)和EsxA-B(r EsxA-B),纯度分别为89%、95%、85%,大小分别为54 ku、33 ku和39 ku。以纯化的r Csa1A、rHlɑ和rEsxA-B 60μg/只分别免疫BALB/c小鼠,共免疫3次,每次间隔14 d,采用间接ELISA法检测各组小鼠免疫后14 d的抗体效价。结果显示,3种重组蛋白免疫后均能刺激小鼠产生较高效价的抗体,三免后抗体效价分别为1:14 700(rCsa1A)、1:16 890(rHlɑ)、1:15 760(rEsxA-B)。利用5型NM2株(2×10^(7)cfu/只)、8型LZ145株(2×10^(7)cfu/只)、336型XJ44株(2×10^(7)cfu/只)3种血清型S. aureus分别经尾静脉注射攻菌,观察并统计各组小鼠的发病和死亡率,评价各重组蛋白的免疫保护率。结果显示,分别经原核表达了rCsa1A、rHlɑ和rEsxAB。攻菌后对照组小鼠全部死亡;3组重组蛋白免疫组96 h内死亡的小鼠在攻菌48 h后出现精神沉郁,食欲不振等症状,而存活小鼠未出现临床症状。r Csa1A、rHlɑ和rEsxA-B免疫组小鼠提供的抵抗NM2、LZ145、XJ44菌株攻击的保护率分别为60%、60%、50%;80%、70%、70%;50%、60%、60%,其中rHlα的免疫保护效果最佳,其对3种攻毒菌株提供的平均保护率为73.3%,是较为理想的亚单位疫苗候选抗原。以上结果表明,S. aureus rHlα能够刺激免疫小鼠产生抗体,对小鼠提供不同程度的保护力。本研究为奶牛乳腺炎S. aureus亚单位疫苗的研制和应用提供参考依据。

To evaluate the immune protective efficacy of Csa1 A, Hlɑ and EsxA-B proteins of Staphylococcus aureus(S. aureus), recombinant plasmids pGEX-4 T-2-CSA1 A, pET-22 B-Hlα and pGEX-4 T-2-ESXA-B were constructed and transformed into E. coli BL21(DE3), respectively in this study. After induction with IPTG and purification by corresponding chromatography column, the expression of recombinant proteins was examined by SDS-PAGE. The results showed that high purity recombinant proteins Csa1 A(rCsa1 A), Hlα(rHlα) and ESXA-B(rEsxA-B) were obtained with purity of 89%, 95% and 85%, and molecular weight of 54 ku, 33 ku and 39 ku, respectively. BALB/c mice were immunized with the purified rCsa1 A, rHlα and rEsxA at the dose of 60μg/mouse, respectively, for 3 times with an interval of 14 days. The indirect ELISA method was used to detect the antibody titer of mice in each group on the 14 th day after immunization. The results showed that all three recombinant proteins stimulated mice to produce high-titer antibodies after immunization, and the antibody titer after the third immunization was1:14700, 1:16890, and 1:15760, respectively. Three serotypes of S. aureus, type 5 NM2 strain(2×10^(7) cfu/mouse), type 8 LZ145 strain(2×10^(7) cfu/mouse), and type 336 XJ44 strain(2×10^(7) cfu/mouse) were injected into the tail vein, respectively. The morbidity and mortality of mice in each group were observed and counted to evaluate the immune protection of the recombinant protein. The results showed that rCsa1 A, rHlα and rEsxA-B were expressed through prokaryotic cells. after bacterial challenge, all mice in the control group died;in the three recombinant protein immunized groups, the mice died within 96 hours showed symptoms such as depression and loss of appetite 48 hours after challenge, while the surviving mice did not show clinical symptoms. The protection rate provided by r Csa1 A against NM2, LZ145 and XJ44 strains was 60%, 60% and 50%, respectively, it was 80%, 70%, 70% for rHlα, and 50%, 60%, 60% for rEsxA-B, of which rHlα had the best immune protection effect with an average protection rate of73.3% against the three challenge strains, making it an ideal candidate antigen for subunit vaccines. The above results show that S. aureus r Hlα can stimulate immunized mice to produce antibodies and provide different degrees of protection to mice, providing a reference for the development and application of S. aureus subunit vaccines for dairy cows.