表达PCV2 ORF2的重组T7噬菌体在猪体内的存留分析

Analysis of the Persistence of Recombinant T7 Phage Expressing PCV2 ORF2 in Pigs

为了检测经颈部肌肉注射入猪体的、表达猪圆环病毒2型(PCV2)衣壳蛋白(ORF2)的重组T7噬菌体(T7-ORF2)在体内的存留情况,本试验将PCV2抗原和抗体均为阴性的20日龄仔猪随机分为试验组和对照组,每组5只;试验组猪只以1.5mL/只剂量颈部肌肉注射增殖收获的T7-ORF2,对照组猪只以相同途径注射等体积的洗脱液;于注射前1 d、注射后10 h、1~7 d测量体温,观察猪只的临床症状和死亡情况;分别于注射后1 h、3 h、5 h、10 h、1 d、3 d、5 d和7 d采集猪只的血液和粪便样品,注射后10 h、1 d、3 d、5 d和7 d采集肝脏、脾脏和腹股沟淋巴结样品;将收集的各样品处理上清与宿主菌(大肠埃希菌BLT5403株)混合培养,通过细菌裂解分析T7-ORF2的感染性;以细菌裂解液提取的基因组DNA为模板进行PCR,检测ORF2基因;通过免疫组织化学检测T7-ORF2在肝脏、脾脏和腹股沟淋巴结中的分布和存留时间。结果显示,试验组猪只在试验期体温、采食、饮水和精神状态正常,无不良症状和死亡发生;注射后5 d能从血清和粪便中检测到感染性的T7-ORF2;注射后3 d能从肝脏、脾脏和腹股沟淋巴结中检测到感染性的T7-ORF2;从细菌裂解液中可扩增检测到PCV2 ORF2基因片段;免疫组织化学切片观察结果显示,T7-ORF2在机体中逐渐被清除,在脾脏中存留至少5 d,在肝脏和腹股沟淋巴结中存留至少7 d。结果表明,T7-ORF2可进入猪只的血液循环、肝脏、脾脏和腹股沟淋巴结中,并随粪便排出体外。本试验为利用T7-ORF2深入开发PCV2新型疫苗提供数据支撑。

To investigate the persistence of recombinant T7 phage(T7-ORF2)expressing the capsid protein(ORF2)of porcine circovirus type 2(PCV2)in pigs after intramuscular injection into the neck,a study was conducted using 20-day-old piglets negative for PCV2 antigen and antibodies.The piglets were randomly divided into Experimental and Control groups,each containing five pigs.The Experimental group received 1.5 mL of proliferated T7-ORF2 by intramuscular injection in the neck,while the Control group received the same volume of eluent via the same route.Body temperature,clinical symptoms,and mortality were monitored one day before injection and at 10 hours,and 1 to 7 days post-injection.Blood and fecal samples were collected at 1 hour,3 hours,5 hours,10 hours,1 day,3 days,5 days,and 7 days post-injection,and liver,spleen,and inguinal lymph node samples were collected at 10 hours,1 day,3 days,5 days,and 7 days post-injection.Samples were mixed with host bacteria(Escherichia coli strain BLT5403)for plaque assays to analyze the infectivity of T7-ORF2.PCR was performed on genomic DNA extracted from bacterial lysates to detect the ORF 2 gene.Immunohistochemistry was used to detect the distribution and persistence of T7-ORF2 in liver,spleen,and inguinal lymph nodes.The results showed that the Experimental group pigs maintained normal body temperature,appetite,water intake,and mental state with no adverse symptoms or mortality during the trial period.Infectious T7-ORF2 was detectable in serum and feces within 5 days post-injection and in liver,spleen,and inguinal lymph nodes within 3 days post-injection.PCV2 ORF 2 gene fragments were amplified from bacterial lysates.Immunohistochemistry indicated that T7-ORF2 was gradually cleared from the body,persisting in the spleen for at least 5 days and in the liver and inguinal lymph nodes for at least 7 days.The study demonstrates that T7-ORF2 can enter the bloodstream,liver,spleen,and inguinal lymph nodes of pigs and be excreted in feces,providing data support for the further development of a novel PCV2 vaccine using T7-ORF2.