【作者】 郑宝亮；

【导师】 童光志；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2006， 博士

【摘要】 猪流感（SI）是一种非常普遍的猪的急性、热性和高度接触性呼吸道系统疾病,其病原主要是H3N2或H1N1亚型猪流感病毒（SIV）。由于猪可感染禽流感病毒和人流感病毒,被认为是流感病毒的混合器和新亚型流感病毒的加工场,因此,研制安全、有效的SIV疫苗来预防猪流感不但可以减少养猪业的经济损失,而且对食品安全和公共卫生具有重要意义。本研究将含有H3N2亚型猪流感病毒A/Swine/Inner Mogolian/547/2001（H3N2）（简称SwIM547）血凝素（HA）基因的转移载体pLTK-HA与伪狂犬病疫苗毒株Bartha-K61的基因组共转染Vero细胞,通过数代蓝斑克隆筛选、纯化后,获得了一株重组伪狂犬病病毒,命名为rPRV-HA。Western-blot实验表明,此重组病毒所表达的HA蛋白的分子量为60ku左右。间接免疫荧光试验显示,HA蛋白表达于被感染细胞的胞浆中。为了评价此重组病毒作为侯选疫苗的免疫效力,本研究先后以鼠和猪为动物模型对其进行了评价。以小鼠为动物模型对重组伪狂犬病病毒rPRV-HA进行的免疫效力评价试验中,按每只10^5.0TCID₅₀ rPRV-HA的剂量通过滴鼻接种8周龄雌性BALB/c小鼠（n=60）,同时设Bartha-K61免疫对照组（n=60）、非免疫攻毒对照组（n=20）和非免疫不攻毒对照组（n=10）。于免疫后不同时间分别从rPRV-HA免疫组和Bartha-K61免疫对照组随机剖杀一定数量的小鼠,其余小鼠于免疫后第28d用10^5.0 TCID₅₀同亚型SIV毒株A/Swine/Heilongjiang/74/2000（H3N2） （SwHLJ74）进行攻击。攻毒后第4、7和14d,分别剖杀小鼠,进行间接免疫荧光、病毒分离、血清学和病理组织学检测。结果表明,重组病毒主要分布于肺脏;免疫后14d起,从rPRV-HA免疫组及Bartha-K61免疫对照组均可检测到针对PRV的荧光抗体;从rPRV-HA免疫组可以检测到针对SIV的荧光抗体和血凝抑制抗体,而各对照组均呈阴性。攻毒后从rPRV-HA免疫组小鼠未分离到攻击病毒,血凝抑制抗体显著升高,病理变化比对照组明显轻微,表明rPRV-HA免疫小鼠可以抵抗同亚型SIV的攻击,可以作为rPRV-HA免疫效力评价模型。以商品猪为动物模型,对重组伪狂犬病病毒rPRV-HA进行的免疫效力评价试验中,按每只10_5.0 TCID₅₀ rPRV-HA的剂量通过肌肉注射接种4周龄断奶仔猪（n=12）,同时设Bartha-K61免疫对照组（n=5）和非免疫攻毒对照组（n=5）。免疫后35d用2×10^5.0 TCID₅₀同亚型SIV毒株A/Swine/Heilongjiang/74/2000（H3N2） （SwHLJ74）进行攻击。免疫组和对照组攻毒后均无临床症状,但对照组有明显的体温升高,显示出了免疫组受到一定的保护作用。小猪鼻拭子分毒结果显示免疫组和对照组在攻毒后6d都能分离到病毒。从支气管和肺泡冲洗液的检测结果可以看出,攻毒后4d各组均可检测到病毒,7d时rPRV-HA免疫组却没有再次检测到病毒,对照组则仍可以检到病毒。rPRV-HA免疫组及Bartha-K61对照组免疫后7d均可检测到针对PRV的中和抗体;rPRV-HA免疫组可以检测到针对SIV的血凝抑制抗体,而各对照组均呈阴性,并且所有对照组小猪也在攻毒之后7d开始产生了血凝抑制（HI）抗体。尸体剖检结果方面,组织冰冻切片的间接免疫荧光未检测到任何病毒粒子,免疫组与对照组均只有个别小猪肺脏出现轻微病理变化,其统计学意义不大,但是显微病理变化方面rPRV-HA免疫组却表现出非常明显的病理保护作用。综合本试验结果,rPRV-HA的免疫接种对同亚型SIV攻击具有较好的保护作用。更多还原

【Abstract】 Influenza is a common respiratory disease in pigs, and it is a acute, feverish and infectious disease. Swine influenza virus（SIV） especially H3N2 and H1N1subtype SIV is the prevalent pathogens of SI. Swine is the only animal which can be infected by either avian or human original influenza viruses, and can be considered as the intermediate host for the process of genetic reassortments between viruses of different hosts.Generate new strains of influenza virus. Vaccination against SIV can reduce economic lose and play a role in food safety and public health.A pseudorabies virus （PRV） transfer vector pLTK-HA containing HA gene of H3N2 SIV was used to cotransfect with genomic DNA of PRV Bartha-K61 into Vero cells. After many cycles of blue plague purification and PCR identification , we got a PRV recombinant containing hemagglutinin （HA） gene of H3N2 SIV and designated as rPRV -HA. Expression of SIV HA by rPRV-HA is about 60ku as demonstrated by Western blotting analysis. Indirect immunofluorescence assay （IFA） demonstrated that HA was expressed by rPRV-HA in cytoplasm.To evaluate the immune efficacy of the recombinant as a candidate vaccine, a mouse model and native host, pigs were used successively in the experimentation. A total of 150 8-week-old female BALB/c mice were used in a mouse model. The mice were each inoculated intranasally with 10^5.0 TCID₅₀ of rPRV-HA （rPRV-HA group, n=60） or attenuated vaccine Bartha-K61 （Bartha-K61group, n=60）, and another two unimmunized groups served as challenged group （n=20） or unchallenged group （n=10）. Some mice in rPRV-HA and Bartha-K61 groups were sacrificed for antigen detection and virus isolation at different days post-immunization （DPI）, and the others were each challenged with 10^5.0 TCID₅₀ of the same subtype SIV 28 DPI. Recombinant virus could be detected in the lungs of rPRV-HA-immunized mice. Antibody responses to PRV were detected by indirect immunofluorescence assay, but not by sero-neutralization test, in both rPRV-HA and Bartha-K61 groups. SIV-specific antibodies were detected by hemagglutination inhibition test only in rPRV-HA group 14 DPI. The rPRV-HA-immunized mice were protected from homologous SIV challenge, as indicated by limiting virus replication and pathological changes of the organs, and boosted antibody responses to SIV post-challenge.A recombinant pseudorabies virus rPRV-HA was also evaluated with a commercial porcine model. One group of 12 four-weeks old healthy piglets was inoculated intramuscularly （i.n.） with 10^5.0 TCID₅₀ of rPRV-HA, and another two control groups （five piglets per group） were mock-inoculated or inoculated with Bartha-K61. Thirty-five days post-inoculation, all piglets were更多还原