【作者】 许家荣；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2010， 博士

【摘要】 1. SIVH1N1和H3N2亚型重组HAl蛋白iELISA方法的建立将合成的SIV H1N1和H3N2的HA1分别插入原核表达载体pET28b(+),构建重组表达质粒pET28b(+)-H1HA1、pET28b(+)-H3HA1。将重组质粒转化大肠杆菌BL21,用IPTG诱导表达,并用镍柱亲和层析法纯化蛋白。用猪流感阳性血清进行Western-blot,证明目的蛋白具有HAl蛋白抗原性。利用SIV HA1重组蛋白作包被抗原,通过反应条件优化,建立了间接ELISA方法用于检测猪流感病毒抗体。结果表明本方法具有较好的特异性、敏感性和重复性,适于大规模检测SIV血清抗体的流行病学调查。2.表达H1N1 HA1重组猪痘病毒的构建与免疫原性分析以质粒pET28b(+)-H1HA1为模板扩增猪流感病毒H1N1 HA1基因,并将其插入猪痘病毒载体pUSZ11中,构建了猪痘病毒转移载体pUSZ11/H1。将pUSZll/H1与猪痘病毒同源重组,获得表达猪流感H1N1 HA1蛋白重组猪痘病毒rSPV/H1。用SⅣH1N1阳性血清进行间接免疫荧光试验(IFA),证明该重组病毒在PK15细胞中可以表达HAl目的蛋白；用Western-blot检测,结果显示HA1蛋白能够正确表达,且抗原性良好。取45只BALB/c小鼠,随机分为3组,每组15只。第1组每只肌肉免疫rSPV/H1,剂量为0.2×1070 TCIDD50;第2组每只肌肉注射wtSPV0.2×107.0TCID5o,第3组每只肌肉注射PK15细胞裂解上清0.2 mL。21 d、35 d分别以相同的剂量加强免疫2次。分别于首次免疫后21、35、42 d采血测定ELISA抗体和中和抗体；在首次免疫后21、35、42d,测定脾淋巴细胞增殖反应；首次免疫后35d,测定脾淋巴细胞培养上清中IFN-γ和IL-4的量。结果为：第1组小鼠在免疫后21d,可以检测到SIV H1N1 HA1特异的ELISA抗体和中和抗体,42d达到最高,抗体水平显著高于对照组(P<0.05)。 21、35、42d,rSPV/H1免疫组小鼠淋巴细胞增殖指数(Stimulation Index, SI)显著高于wtSPV、PK15细胞免疫组(P<0.05)；35d时,rSPV/H1免疫组小鼠脾淋巴细胞培养上清中IFN-γ(175.68 pg/ml)和IL-4(117.4 pg/ml)平均含量显著高于对照组(P<0.05)。取雌性Hartly豚鼠36只,随机分为6组,每组6只。第1、2组每只免疫rSPV/H1,剂量为每只0.4×107.0TCID50;第3、4组每只免疫wtSPV,剂量为0.4×107.0TCID50;第5、6组每只注射PK15细胞裂解上清0.4 mL。21 d加强免疫1次。首次免疫后28 d,测定外周血淋巴细胞培养上清中IFN-Y和IL-4的量。35 d测定中和抗体滴度。第35 d攻毒,第1、3、5组每只鼻腔注射0.2×105.0TCID50的HN1 SIV; 2、4、6组每只鼻腔注射0.2×105.0TCID50的H3N2 SIV。攻毒后观察7 d。对临床症状和肺大体病变进行评分。42 d将全部豚鼠安乐死,取肺组织处理、接种SPF鸡胚,HI试验检测流感病毒,同时观察肺组织病理变化。结果显示,免疫后28 d,1、2组淋巴细胞培养上清中SIV H1N1特异的的细胞因子IFN-γ(257.3 pg/ml)和IL-4 (208.26 pg/ml)的量显著高于对照组(P<0.05)。免疫35d, rSPV/Hl免疫组豚鼠血清中均能够检测到SⅣH1N1特异的中和抗体,而wtSPV、PK15免疫组血清对SIV H1N1没有中和活性。一免后35 d攻毒。攻毒后,免疫rSPV/H1的豚鼠无流感症状出现,而对照组豚鼠则表现有流感症状。rSPV/H1免疫组中只有用SIV H3N2攻击的2/6只豚鼠有轻微肉眼可见病理变化,而所有对照组豚鼠肺部都有范围大小不等的紫色、肉变区出现,病变范围从35%-75%。显微镜下见有严重的间质增生,有的充血、淋巴细胞浸润和肺泡壁破坏。免疫rSPV/H1的豚鼠只从用SIV H3N2攻击的1/6只中分离到SⅣ,而对照组全都分离到SⅣ。取30头6周龄SIV、PRRSV、PCV、SPV阴性猪,随机分6组,每组5头。第1组和第2组免疫rSPV/H1,第3、4组注射wtSPV,第5、6组注射PK15细胞裂解上清。在免疫后21d,检测H1N1的中和抗体和外周血淋巴细胞培养上清中IFN-Y和IL-4的量。在免疫后21d,所有猪攻毒。第1、3、5组每只鼻腔注入1×105.0 TCID50的HN1；2、4、6组每只鼻腔注入1×105.0 TCID50的H3N2。攻毒后连续5 d,观察临床表现,测体温、采鼻拭子。剖检时记录肺大体病变。检测肺组织SⅣ；同时取肺组织做病理切片。结果显示,免疫后21 d,1、2组淋巴细胞培养上清中的细胞因子IFN-γ (247.48pg/ml)和IL-4(159.02 pg/ml)的量显著高于对照组(P<0.05). rSPV/Hl免疫组猪血清中均能够检测H1N1特异的中和抗体,wtSPV,PK15免疫组血清对SIV H1N1没有中和活性。攻毒后,免疫rSPV/H1的猪均无流感症状出现,而对照组则表现有流感症状。PK15和wtSPV组猪的鼻腔中一直持续分泌SIV,而重组毒免疫组在1、2天有2头检测到病毒,且分泌量明显低于对照组；其他猪未检测到病毒。rSPV/Hl免疫组猪中,用H3N2攻击的3/5头猪有轻微肉眼可见病理变化,而所有对照组猪肺部都有3%-9%的病变。免疫rSPV/Hl的猪只从用H3N2攻击的1/5号猪分离到H3N2,而对照组猪全都分离到H3N2。上述结果表明：本研究获得了SIV H1N1 HA1蛋白重组猪痘病毒rSPV/H1。 rSPV/H1能诱导小鼠产生显著的体液免疫和细胞免疫反应,不仅保护豚鼠和猪抵御同源SIV H1N1的攻击,还能部分抵御异型SIV H3N2的攻击。3.表达H3N2 HA1重组猪痘病毒的构建与免疫原性分析以质粒pET28b(+)-H3HA1为模板扩增H3N2 HA1基因,并将其插入猪痘病毒载体pUSZ11中,构建了猪痘病毒转移载体pUSZ11/H3。将pUSZ11/H3与猪痘病毒同源重组,获得表达H3N2 HA1蛋白重组猪痘病毒rSPV/H3。用H3N2阳性血清进行间接免疫荧光试验(IFA),证明该重组病毒在PK15细胞中可以表达HA1目的蛋白；用Western-blot检测,结果显示HA1蛋白能够正确表达,且抗原性良好。取45只BALB/c小鼠,随机分为3组,每组15只。第1组每只肌肉免疫rSPV/H3,第2组每只肌肉注射wtSPV,第3组每只肌肉注射PK15细胞裂解上清。21 d、35 d分别以相同的剂量加强免疫2次。分别于首次免疫后21d、35d、42 d采血测定ELISA抗体和中和抗体；在首次免疫后21d、35d、42d,测定脾淋巴细胞增殖反应；首次免疫后35d,测定脾淋巴细胞培养上清中SIV H3N2特异的IFN-γ和IL-4的量。结果为：第1组小鼠在免疫后21d,可以检测到H3N2 HA1特异的ELISA抗体和中和抗体,42d达到最高,抗体水平显著高于对照组(P<0.05)。21d、35d、42d,rSPV/H3免疫组小鼠脾淋巴细胞增殖指数显著高于wtSPV、PK15细胞免疫组(P<0.05);35d时,rSPV/H3免疫组小鼠脾淋巴细胞培养上清中IFN-γ(188.82pg/ml)和IL-4(133.57pg/ml)平均含量显著高于对照组(P<0.05)。取雌性Hartly豚鼠36只,随机分为6组,每组6只。第1、2组每只免疫rSPV/H3,第3、4组每只免疫wtSPV,第5、6组每只注射PK15细胞裂解上清。21d加强免疫1次。首次免疫后28 d,测定外周血淋巴细胞培养上清中IFN-γ和IL-4的量。35 d测定中和抗体滴度。第35 d攻毒,第1、3、5组每只鼻腔注射SIV H3N2; 2、4、6组每只鼻腔注射SIVH1N1。攻毒后观察7 d。记录临床症状和肺大体病变。42 d将全部豚鼠安乐死,取肺组织处理、接种SPF鸡胚,HI试验检测流感病毒,同时取肺组织固定,做病理切片。结果显示,免疫后28 d,1、2组淋巴细胞培养上清中SIV H3N2特异的的细胞因子IFN-y(253.26pg/ml)和IL-4(191.88pg/ml)的量显著高于对照组(P<0.05)。免疫35d,rSPV/H3免疫组豚鼠血清中均能够检测到H3N2特异的中和抗体,wtSPV.PK15免疫组血清对H3N2没有中和活性。一免后35 d攻毒。攻毒后,免疫rSPV/H3的豚鼠无流感症状出现,而对照组豚鼠则表现有流鼻涕等流感症状。rSPV/H3免疫组中只有用H1N1攻击的有2只有轻微肉眼可见病理变化,而所有对照组豚鼠肺部都有大范围大小不等的紫色、肉变区出现,病变范围从35%-75%。显微镜下见有严重的间质增生,有的充血、淋巴细胞浸润和肺泡壁破坏。免疫rSPV/H3的豚鼠只从用H1N1攻击的1/6只的肺中分离到H1N1,而对照组全都分离到H1N1 SIV。取30头6周龄SIV(H3N2、H1N1)、PRRSV、PCV2、SPV阴性猪,随机分6组,每组5头。第1组和第2组免疫rSPV/H3,第3、4组注射wtSPV,第5、6组注射PK15细胞裂解上清。在免疫后21d,检测SIV的中和抗体和外周血淋巴细胞培养上清中H3N2特异的IFN-y和IL-4的量。在免疫后21d,所有猪攻毒。第1、3、5组每只鼻腔注入1×105.0TCID50的H3N2；2、4、6组每只鼻腔注入1×105.OTCID50的H1N1。攻毒后连续5 d,观察临床表现,测体温、采鼻拭子。剖检时记录肺大体病变。检测肺组织SIV；同时取肺组织做病理切片。结果显示,免疫后21 d,1、,2组淋巴细胞培养上清中的细胞因子IFN-γ(264.10pg/ml)和IL-4(180.16pg/ml)的量显著高于对照组(P<0.05)。rSPV/H3免疫组猪血清中均能够检测到SⅣH3N2特异的中和抗体,wtSPV、PK15免疫组血清对SIV H3N2没有中和活性。攻毒后,免疫rSPV/H3的猪无流感症状出现,而对照组则表现有流感症状。PK15和wtSPV组猪的鼻腔中SⅣ分泌一直持续,而重组毒免疫组用异型SIV H1N1攻击的2/5头检出病毒,且分泌量明显低于对照组；其他猪未检测到病毒。rSPV/H3免疫组猪中,用H1N1攻击的有2头有轻微肉眼可见病理变化,而所有对照组猪肺部都有3%-9%的病变。免疫rSPV/H3的猪未分离到H1N1,而对照组猪全都分离到H1N1。上述结果表明：本研究获得了H3N2 HAl蛋白重组猪痘病毒rSPV/H3. rSPV/H3能诱导小鼠产生显著的体液免疫和细胞免疫反应,不仅保护豚鼠和猪抵御同源H3N2的攻击,还能部分抵御异型H1N1的攻击。4.串联表达H3和H1亚型的HA1重组猪痘病毒的构建与免疫原性分析本研究设计、合成基因H3-2A-H1(HA1 Of SIV H3N2,2A of FMDV and HAl gene Of SIV H1N1),将其插入猪痘病毒载体pUSZ11,获得pUSZ11/H3-2A-H1重组猪痘病毒转移载体。将pUSZ11/H3-2A-H1与猪痘病毒同源重组,获得表达猪流感H3N2和H1N1 HA1蛋白的重组猪痘病毒rSPV/H3-2A-H1。用H3N2和H1N1阳性血清进行间接免疫荧光试验(IFA),证明该重组病毒在PK15细胞中可以表达H3N2和H1N1 HA1目的蛋白；用Western-blot检测,结果显示H3N2和H1N1 HA1蛋白能够正确表达,且抗原性良好。取45只BALB/c小鼠,随机分为3组,每组15只。第1组每只肌肉免疫rSPV/H3-2A-H1,剂量为0.2×107.0 TCID50;第2组每只肌肉注射wtSPV 0.2×107.0 TCID50,第3组每只肌肉注射PK15细胞裂解上清0.2 mL。21 d、35 d分别以相同的剂量加强免疫2次。分别于首次免疫后21d、35d、42 d采血测定ELISA抗体和中和抗体；在首次免疫后21d、35d、42d,测定脾淋巴细胞增殖反应；首次免疫后35d,测定脾淋巴细胞培养上清中IFN-γ和IL-4的量。结果为：第1组小鼠在免疫后21d,可以检测到SIV H3N2和H1N1 HA1特异的ELISA抗体和中和抗体,42d达到最高,抗体水平显著高于对照组(P<0.05)。21d、35d、42d,rSPV/H3-2A-H1免疫组小鼠淋巴细胞增殖指数显著高于wtSPV、PK15细胞免疫组(P<0.05);35d时,rSPV/H3-2A-H1免疫组小鼠脾淋巴细胞培养上清中IFN-γ和IL-4平均含量显著高于对照组(P<0.05)。取雌性Hartly豚鼠36只,随机分为6组,每组6只。第1、2组每只免疫rSPV/H3-2A-H1,剂量为每只0.4×107.0 TCID50;第3、4组每只免疫wtSPV,剂量为0.4×107.0 TCID50;第5、6组每只注射PK15细胞裂解上清0.4 mL。21 d加强免疫1次。首次免疫后28 d,测定外周血淋巴细胞培养上清中IFN-γ和IL-4的量。35 d测定中和抗体滴度。第35 d攻毒,第1、3、5组每只鼻腔注射0.2×105.0 TCID50的H3N2 SIV; 2、4、6组每只鼻腔注射0.2×105.0TCID50的H1N1 SIV。攻毒后观察7 d。记录临床症状和肺大体病变。42 d将全部豚鼠安乐死,取肺组织处理、接种SPF鸡胚,HI试验检测流感病毒,同时取肺组织固定、做病理切片。结果为：免疫后28 d,1、2组淋巴细胞培养上清中SIV特异的的细胞因子IFN-γ和IL-4的量显著高于对照组(P<0.05)。免疫35d,rSPV/H3-2A-H1免疫组豚鼠血清中均能够检测到SIV H3N2和H1N1特异的中和抗体,wtSPV、PK15免疫组血清对SⅣ没有中和活性。一免后35 d攻毒。攻毒后,免疫rSPV/H3-2A-H1的豚鼠无流感症状出现,而对照组豚鼠则表现流鼻涕等流感症状。rSPV/H3-2A-H1免疫组无肉眼可见的肺部病理变化,而对照组豚鼠肺部都有范围大小不等的紫色、肉变区出现,病变范围从35%-75%。显微镜检查见有严重的间质增生,有的充血、淋巴细胞浸润和肺泡壁破坏。免疫rSPV/H3-2A-H1的豚鼠未分离到SⅣ,而对照组全都分离到SⅣ。取30头6周龄SIV、PRRSV、PCV、SPV阴性猪,随机分6组,每组5头。第1组和第2组免疫rSPV/H3-2A-H1,第3、4组注射wtSPV,第5、6组注射PK15细胞裂解上清。在免疫后21d,检测SIV H3N2和H1N1的中和抗体和外周血淋巴细胞培养上清中SIV特异的IFN-y和IL-4的量。在免疫后21d,所有猪攻毒。第1、3、5组每只鼻腔注入1×105.0 TCID50的SIV H3N2;2、4、6组每只鼻腔注入1×105.0TCID50的SIV H1N1。攻毒后连续5d,观察临床表现,测体温、采鼻拭子。剖检时记录肺大体病变。检测肺组织SIV；同时取肺组织做病理切片。结果显示,免疫后21d,1、2组淋巴细胞培养上清中的细胞因子IFN-y和IL-4的量显著高于对照组(P<0.05)。rSPV/H3-2A-H1免疫组猪血清中均能够检测到H3N2和H1N1特异的中和抗体,wtSPV、PK15免疫组血清对SIV没有中和活性。攻毒后,免疫rSPV/H3-2A-H1的猪无流感症状出现,而对照组有流感症状。PK15和wtSPV组猪的鼻腔中有SIV分泌,重组毒免疫组未检测到病毒。rSPV/H3-2A-H1免疫组猪中,无肉眼可见病理变化,而所有对照组猪肺部都有3%-9%的病变。从免疫rSPV/ H3-2A-H1的猪肺未分离到SIV,而对照组猪全都分离到SIV。上述结果表明,本研究获得了串联表达H3N2和H1N1 HA1蛋白重组猪痘病毒rSPV/H3-2A-H1。rSPV/H3-2A-H1能诱导小鼠产生显著的体液免疫和细胞免疫反应,能完全保护豚鼠和猪抵御同源H3N2和H1N1的攻击。更多还原

【Abstract】 1. Establishment of an indirect ELISA for detecting antibodies against H1N1 and H3N2 subtype swine influenza virusTo get the recombinant HA1 protein for detection of the antibody titer of SIV H1N1 and H3N2, the HA1 genes of SIV was synthesized and inserted into prokaryotic expression vector pET28b(+). Then the fusion protein was highly expressed in E.coli BL21 induced by IPTG. It could be purified efficiently with Ni+ affinity chromatography column. Western-blot analysis showed that the fusion protein was able to react with swine polyclonal antibody against swine influenza virus H1N1 and H3N2. It indicated that the recombinant protein had the immunogenicity of SIV. The successful expression and identification of the recombinant protein provide the basis for detection the antibody titer of SIV.An indirect ELISA was performed to detect antibodies to swine influenza virus with recombinant HA1 protein. The recombinant antigen had no cross reaction with the antibodies against Hog cholera virus (HCV), porcine circovirus 2(PCV2), foot and mouth disease virus(FMDV), porcine reproductive and respiratory syndrome virus (PRRSV).The results indicated that the indirect ELISA is not only sensitive and specific, but also suitable for large-scale epidemiological investigation for SIV infection.2. Construction and immunogenic analysis of recombinant swine poxvirus expressing HA1 gene of H1N1 subtype SIVSIV H1N1 HA1 gene was amplified from pET28b(+)-HlHA1 by PCR. And then it was inserted into the swine poxvirus shuttle vector. The recombinant plasmid was identified by PCR, restriction enzyme digestion and then sequenced, named as pUSZl1/H1. The swine poxvirus incubated PK15 cell, and then pUSZ11/H1 were transfected. At 7 days post incubation, the recombinant swine poxvirus came up with CPE. Then, the expressions of these target proteins were identified by IFA and western blot; it indicated that a recombinant swine poxvirus, rSPV/H1 expressing the HA1 protein of SIV H1N1 was successfully constructed.Forty-five BALB/c mice were randomly separated into 3 groups with 15 in each. The mice in the first group were immunized with rSPV/H1 (expressing HA1 protein). Group 2 and Group 3 were ranged as control. Group 2 were immunized with wtSPV and Group 3 were immunized with PK15, respectively. After 3 weeks and 5 weeks, the all groups were boosted vaccined except those mice were slaughtered. And SIV-specific antibody, lymphocyte proliferation index, IFN-y and IL-4 were detected to evaluate the immunization at different time after vaccination. The results showed that the antibody against SIV became detectable in group1 at 21 days post immunization (dpi), and reached to the peak at 42dpi. The lymphocytes proliferation index in group 1 was significant higher than that of the control. The secretion of IFN-y and IL-4 of the lymphocytes from the mice immunized with rSPV/H1 is significant higher than that with wtSPV and PK15. These results indicated that the recombinant swine poxvirus rSPV/Hl could induce distinctly higher humoral and cell immune response than wtSPV and PK15 in mice.Thirty-six female Hartly guinea pigs were randomly separated into 6 groups with 6 in each. The first two groups were immunized with the recombinant swine poxvirus rSPV/H1 (0.4×107.0CID50/mL), respectively. And the others as control, group 3 and group 4 were immunized with wtSPV (0.4×107.0TCID50/mL), group 5 and group 6 were immunized with PK15 (0.4mL), respectively. At 21 dpi, the all groups were boosted vaccined. At 35 dpi, group 1,3,5 were challenged with SIV H1N1 and group 2,4,6 were challenged with SIV H3N2. Then clinical signs, SIV specific antibody, IFN-y and IL-4 were detected at different time after vaccination. At 42 dpi, gross tissue lesion and microscope lesion were used to judge the protective efficacy. The results showed that at 35 dpi, rSPV/H1 immunized guinea pigs developed significant higher level of SIV-specific antibodies compared with the control. The highest NA titer in rSPV/Hl group was 1:64. The contents of IFN-y and IL-4 were 257.38pg/ml and 208.26pg/ml in rSPV/H1 immunized guinea pigs, which are significantly higher than that immunized with wtSPV and PK15 at 28 dpi. The guinea pigs immunized with rSPV/H1 showed no clinical signs and the control groups showed significant clinical signs. No SIV were isolated from group 1 and in group 2 SIV were isolated from one guinea pig. However, SIV were isolated from all guinea pigs in control groups. Meanwhile, the guinea pigs in control groups experienced the most serious tissue lesion and only two guinea pigs in group 2 showed slight lung lesion. All results indicated rSPV/H1 could provide not only a protection against SIV H1N1 challenge but also partly a protection against SIV H3N2 challenge in guinea pigs.Thirty 6-week old commercial pigs free of SIV, PRRSV, PCV2 and SPV were randomly separated into 6 groups with 5 in each. The first two groups were immunized with the recombinant swine poxvirus rSPV/H1 (1×107.0TCID50/mL), respectively. And the others as control,group 3 and group 4 were immunized with wtSPV (1×107.0TCID50/mL), group 5 and group 6 were immunized with PK15(1mL), respectively. At 21 dpi, group 1,3, 5 were challenged with SIV HlN1 and group 2,4,6 were challenged with SIV H3N2. Then clinical signs, the rectal temperature, nasal shedding, SIV specific antibody, IFN-y and IL-4 were detected at different time after vaccination. At 26 dpi, gross tissue lesion and microscope lesion were used to judge the protective efficacy. The results showed that at 21dpi, rSPV/H1 immunized pigs developed significant higher level of SIV-specific antibodies compared with the control. The highest NA titer in rSPV/H1 group was 1:32. The contents of IFN-y and IL-4 were 247.48pg/ml and 159.02pg/ml in rSPV/Hl immunized pigs, which are significantly higher than that immunized with wtSPV and PK15. The pigs immunized with rSPV/H1 showed no clinical signs and the control groups showed clinical sign. The pigs immunized with rSPV/Hl showed the lack and decrease of nasal shedding of virus following challenge.No SIV were isolated from lungs group 1 and in group 2 SIV were isolated from one piglet. However, SIV were isolated from all piglet lungs in control groups. Meanwhile, the piglets in control groups experienced distinct tissue lesion. All results indicated rSPV/H1 could provide not only a protection against SIV HlN1 challenge but also partly a protection against SIV H3N2 challenge in piglets.In summary, a recombinant swine poxvirus, rSPV/H1 expressing the HA1 protein of SIV (H1N1) was successfully constructed and identified. rSPV/H1 could induce distinctly higher humoral and cell immune response in mice, guinea pigs and piglets. All results indicated rSPV/H1 could provide not only a protection against SIV H1N1 challenge but also partly a protection against SIV H3N2 challenge in guinea pigs and pigs.3. Construction and immunogenic analysis of recombinant swine poxvirus expressing HA1 gene of H3N2 subtype SIVH3N2 subtype SIV HA1 gene was amplified from pET28b(+)-H3HA1 by PCR. And then it was inserted into the swine poxvirus vector. The recombinant plasmid was identified by PCR, restriction enzyme digestion and then sequenced, named as pUSZll/H3. The swine poxvirus incubated PK15 cell, and then pUSZ11/H3 were transfected. At 7 days post incubation, the recombinant swine poxvirus came up with CPE. Then, the expressions of these target proteins were identified by IFA and western blot; it indicated that a recombinant swine poxvirus, rSPV/H3 expressing the HA1 protein of SIV H3N2 was successfully constructed.Forty-five BALB/c mice were randomly separated into 3 groups with 15 in each. The mice in the first group were immunized with rSPV/H3. Group 2 and Group 3 were ranged as control. Group 2 were immunized with wtSPV and Group 3 were immunized with PK15 cell supernatant, respectively. After 3 weeks and 5 weeks, the all groups were boosted vaccined except those mice were slaughtered. And SIV-specific antibody, lymphocyte proliferation index, IFN-y and IL-4 were detected to evaluate the immunization at different time after vaccination. The results showed that the antibody against SIV H3N2 became detectable in groupl at 21 days post immunization (dpi), and reached to the peak at 42dpi. The lymphocytes proliferation index in group 1 was significant higher than that of group 2 and group 3. The secretion of IFN-y(188.82pg/mL)and IL-4(133.57 pg/mL)of the lymphocytes from the mice immunized with rSPV/H3 is significant higher than that with wtSPV and PK15. These results indicated that the recombinant swine poxvirus rSPV/H3 could induce distinctly higher humoral and cell immune response than wtSPV and PK15 in mice.Thirty-six female Hartly guinea pigs were randomly separated into 6 groups with 6 in each. The first two groups were immunized with the recombinant swine poxvirus rSPV/H3, respectively. And the others as control, group 3 and group 4 were immunized with wtSPV, group 5 and group 6 were immunized with PK15, respectively. At 21 dpi, the all groups were boosted vaccined. At 35 dpi, group 1,3,5 were challenged with H3N2 subtype SIV and group 2,4,6 were challenged with H1N1 subtype SIV. Then clinical signs, SIV specific antibody, IFN-γ and IL-4 were detected at different time after vaccination. At 42 dpi, gross tissue lesion and microscope lesion were used to judge the protective efficacy. The results showed that at 35 dpi, rSPV/H3 immunized pigs developed significant higher level of SIV-specific antibodies compared with the control. The highest NA titer in rSPV/H3 group was 1:64. The contents of IFN-y and IL-4 were 253.26pg/ml and 191.88pg/ml in guinea pigs immunized with rSPV/H3, which are significantly higher than that immunized with wtSPV and PK15 at 28 dpi. The guinea pigs immunized with rSPV/H3 showed no clinical signs and the control groups showed significant clinical signs. No SIV were isolated from group 1 and SIV were isolated from one guinea pig in group 2. However, SIV were isolated from all guinea pigs in control groups. Meanwhile, the guinea pigs in control groups experienced the most serious tissue lesion, but only two guinea pigs in group 2 showed slight lung lesion. All results indicated rSPV/H3 could provide not only a protection against H3N2 subtype SIV challenge but also partly a protection against H1N1 subtype SIV challenge in guinea pigs.Thirty 6-week old commercial pigs free of SIV, PRRSV, PCV2 and SPV were randomly separated into 6 groups with 5 in each. The first two groups were immunized with the recombinant swine poxvirus rSPV/H3, respectively. And the others as control, group 3 and group 4 were immunized with wtSPV, group 5 and group 6 were immunized with PK15, respectively. At 21 dpi, group 1,3,5 were challenged with H3N2 subtype SIV and group 2,4,6 were challenged with H1N1 subtype SIV. Then clinical signs, the rectal temperature, nasal shedding, SIV specific antibody, IFN-γ and IL-4 were detected at different time after vaccination. At 26 dpi, gross tissue lesion and microscope lesion were used to judge the protective efficacy. The results showed that at 21dpi, rSPV/H3 immunized pigs developed significant higher level of SIV-specific antibodies compared with the control. The highest NA titer in rSPV/H3 group was 1:32. The contents of IFN-y and IL-4 were 264.10pg/ml and 180.16pg/ml in rSPV/H3 immunized pigs, which are significantly higher than that immunized with wtSPV and PK15. The pigs immunized with rSPV/H3 showed no clinical signs and the control groups showed clinical sign. The pigs immunized with rSPV/H3 showed the lack and decrease of nasal shedding of virus following challenge.No SIV were isolated from lungs group 1 and group 2. However, SIV were isolated from all piglets lungs in control groups. Meanwhile, the piglets in control groups experienced tissue lesion. All results indicated rSPV/H3 could provide not only a protection against SIV H3N2 challenge but also partly a protection against SIV H1N1 challenge in piglets.In summary, a recombinant swine poxvirus, rSPV/H3 expressing the HA1 protein of SIV (H3N2) was successfully constructed and identified. rSPV/H3 could induce distinctly higher humoral and cell immune response in mice, guinea pigs and piglets. All results indicated rSPV/H3 could provide not only a protection against SIV H3N2 challenge but also partly a protection against SIV H1N1 challenge in guinea pigs and pigs.4. Construction and immunogenic analysis of recombinant swine poxvirus co-expressing HA1 gene of H3N2 and H1N1 subtype SIVTo get the recombinant swine poxvirus co-expressing HA1 gene of H3N2 and HlN1 subtype SIV, H3-2A-H1 gene was designed, synthesized and inserted into swine poxvirus shuttle vector pUSZ11. The recombinant plasmid was identified by PCR, restriction enzyme digestion and then sequenced, named as pUSZll/H3-2A-Hl. The swine poxvirus incubated PK15 cell, and then pUSZll/H3-2A-H1 were transfected. At 7 days post incubation, the recombinant swine poxvirus came up with CPE. Then, the expressions of these target proteins were identified by IFA and western blot; it indicated that a recombinant swine poxvirus, rSPV/H3-2A-Hl expressing the HA1 protein of H3N2 and H1N1 subtype SIV was successfully constructed.Forty-five BALB/c mice were randomly separated into 3 groups with 15 in each. The mice in the first group were immunized with rSPV/H3-2A-H1. Group 2 and Group 3 were ranged as control. Group 2 were immunized with wtSPV and Group 3 were immunized with PK15, respectively. After 3 weeks and 5 weeks, the all groups were boosted vaccined except those mice were slaughtered. And SFV-specific antibody, lymphocyte proliferation index, IFN-y and IL-4 were detected to evaluate the immunization at different time after vaccination. The results showed that the antibody against SIV became detectable in group 1 at 21 days post immunization (dpi), and reached to the peak at 42dpi. The lymphocytes proliferation index in group 1 was significant higher than that of the control. The secretion of IFN-y and IL-4 of the lymphocytes from the mice immunized with rSPV/H3-2A-H1 is significant higher than that with wtSPV and PK15.These results indicated that the recombinant swine poxvirus rSPV/H3-2A-H1 could induce distinctly higher humoral and cell immune response than wtSPV and PK15 in mice.Thirty-six female Hartly guinea pigs were randomly separated into 6 groups with 6 in each. The first two groups were immunized with the recombinant swine poxvirus rSPV/H3-2A-H1 (0.4×107.OTCID5o/mL), respectively. And the others as control, group 3 and group 4 were immunized with wtSPV (0.4x107.0TCID50/mL), group 5 and group 6 were immunized with PK15 (0.4mL), respectively. At 21 dpi, the all groups were boosted vaccined. At 35 dpi, group 1,3,5 were challenged with SIV H1N1 and group 2,4,6 were challenged with SIV H3N2. Then clinical signs, SIV specific antibody, IFN-y and IL-4 were detected at different time after vaccination. At 42 dpi, gross tissue lesion and microscope lesion were used to judge the protective efficacy. The results showed that at 35 dpi, rSPV/H3-2A-Hl immunized pigs developed significant higher level of SIV-specific antibodies compared with the control. The contents of IFN-y and IL-4 were significantly higher than that immunized with wtSPV and PK15 at 28 dpi in guinea pigs immunized with rSPV/H3-2A-H1. The guinea pigs immunized with rSPV/H3-2A-H1 showed no clinical signs and the control groups showed significant clinical signs. No SIV were isolated from group 1 and group 2. However, SIV were isolated from all guinea pigs in control groups. Meanwhile, in group 1 and group 2,the guinea pigs did not show tissue lesion,but in control groups experienced the most serious tissue lesion. All results indicated rSPV/H3-2A-H1 could provide a total protection against H1N1 and H3N2 subtype SIV challenge in guinea pigs.Thirty 6-week old commercial pigs free of SIV, PRRSV, PCV2 and SPV were randomly separated into 6 groups with 5 in each. The first two groups were immunized with the recombinant swine poxvirus rSPV/H3-2A-H1(1×107.0CID50/mL), respectively. And the others as control, group 3 and group 4 were immunized with wtSPV (1×107.0TCID50/mL), group 5 and group 6 were immunized with PK15 (1mL), respectively. At 21 dpi, group 1,3,5 were challenged with SIV H1N1 and group 2,4,6 were challenged with SIV H3N2. Then clinical signs, the rectal temperature, nasal shedding, SIV specific antibody, IFN-γ and IL-4 were detected at different time after vaccination. At 26 dpi, gross tissue lesion and microscope lesion were used to judge the protective efficacy. The results showed that at 21dpi, pigs immunized with rSPV/H3-2A-H1 developed significant higher level of SIV-specific antibodies compared with the control. In rSPV/H3-2A-H1 immunized pigs the contents of IFN-γ and IL-4 were significantly higher than that immunized with wtSPV and PK15. The pigs immunized with rSPV/H3-2A-H1 showed no clinical signs and the control groups showed clinical sign. The pigs immunized with rSPV/H3-2A-H1 showed the lack of nasal shedding of virus following challenge.No SIV were isolated from lung group 1 and group 2. However, SIV were isolated from all piglets lung in control groups. Meanwhile, the piglets in control groups experienced distinct tissue lesion. All results indicated rSPV/H3-2A-H1 could provide a protection against H1N1 and H3N2 subtype SIV challenge in piglets.In summary, a recombinant swine poxvirus, rSPV/H3-2A-H1 co-expressing the HA1 protein of SIV H3N2 and H1N1 was successfully constructed and identified. rSPV/H3-2A-H1 could induce distinctly higher humoral and cell immune response in mice, guinea pigs and piglets. All results indicated rSPV/H3-2A-H1 could provide a total protection against H1N1 and H3N2 subtype SIV challenge in guinea pigs and pigs.更多还原