鸡源鹦鹉热衣原体主要外膜蛋白（MOMP）基因重组腺病毒活载体疫苗研究
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摘要
本研究分离了鸡源鹦鹉热衣原体,建立了鸡衣原体病PCR诊断方法。利用E1和E3缺失的人腺病毒5型载体,首次在国内外成功构建了含有鸡源鹦鹉热衣原体主要外膜蛋白(MOMP)基因的重组腺病毒。并用重组腺病毒对本动物鸡进行了免疫试验和安全试验。
(1) 从疑似患鹦鹉热衣原体病鸡分离到衣原体,经涂片观察、接种鸡胚,碘染色试验、磺胺嘧啶钠试验、电子显微镜观察,确定为鹦鹉热衣原体,命名为hnq株,用鸡胚法测定其毒力为1.8×10~(-7)/0.2ELD_(50),CpL株的毒力为5.6×10~(10)/0.2 ELD_(50)。选择CpL株为研究对象。建立了鸡源鹦鹉热衣原体PCR诊断方法,并对其特异性、敏感性和灵敏度进行了验证。
(2) 对鸡源鹦鹉热衣原体CpL株MOMP基因进行了克隆和测序,并与禽源鹦鹉热衣原体其他菌株的MOMP基因进行了序列比较。结果表明,与禽源衣原体鸭(GD)株的MOMP氨基酸序列同源性为89.7%;与火鸡(CT1)株MOMP氨基酸序列同源性为88.7%;与鸽子(CP3)株MOMP氨基酸序列同源性为82.3%;与长尾小鹦鹉(VS225)株的MOMP氨基酸序列同源性为94.4%。
(3) 通过基因操作的方法,将鸡源鹦鹉热衣原体CpL株MOMP基因置于腺病毒穿梭载体的巨细胞瘤(CMV)启动子和SV40 Poly(A)尾巴之间,构建成目的基因的表达盒。构建的穿梭载体与腺病毒骨架载体在BJ5183工程菌内进行同源重组,筛选阳性重组腺病毒质粒,命名为Ad-MOMP。序列测定表明,Ad-MOMP重组腺病毒所带的鸡源鹦鹉热衣原体MOMP基因的氨基酸序列完全正确。转染HEK293细胞,用间接免疫荧光对表达产物检测,证明所构建的重组腺病毒能正确表达目的基因。减蛋综合征(EDS_(76))阳性血清中和试验表明,不能中和重组腺病毒。重组腺病毒稳定性试验表明,该重组病毒能稳定携带外源基因进行传代(23代)。
(4) 禽源鹦鹉热衣原体MOMP基因重组腺病毒免疫SPF小鸡,第21天测定抗MOMP抗体,达到1:32,用6.8×10~9TCID_(50)免疫SPF小鸡。能够抵抗5.6×10~10ELD_(50)同源强毒CpL株的攻击获得保护,保护率达9/10;用减蛋综合征病毒(EDS_(76)V)检测重组腺病毒抗体,表明二者无抗原—抗体交叉反应。用野生型腺病毒和HEK293细胞免疫的对照组均未产生任何的免疫保护效果。肌肉与皮下两种免疫途径试验结果表明,二者基本无差别,考虑到田间免疫的实用性,选择肌肉免疫。
(5)根据SPF小鸡免疫攻毒试验结果,用6.8×10~9TCID_(50)重组腺病毒一次肌肉免疫7日龄小鸡,21天后用同源强毒CpL株进行攻击,观察结果6个月,表明免疫小鸡能够抵抗1.1×10~10ELD_(50)鹦鹉热衣原体强毒的攻击,保护率达8/10。对照组10只鸡剖检后全部表现出鹦鹉热衣原体症状。抗体持续期试验表明,免疫后21天抗体水平达到最高(1:64),随后开始逐渐下降,至180天时,抗体水平仍然维持在保护水平。应用衣原体IHA和四甲基偶氮唑盐(MTT)法检测了小鸡的体液免疫和细胞免疫,表明二者都有应答。重组腺病毒安全性试验表明,小鸡生长正常,无排毒现象。重组腺病毒保存期试验显示,-20℃保存1年后 该重组腺病毒滴度有所下降,但不影响免疫力。
A chlamydia psittaci were isolated from affected chickens and the PCR method for chicken chlamydiosis was established. Recombinant adenovirus containing major outer membrane protein (MOMP) gene of Chlamydia psittaci of chicken origin were successfully constructed with human adenovirus type 5 vector which lack El and E3 genes. The MOMP genes were expressed in HEK293 cells and the recombinant adenovirus were evaluated in vitro and in vivo. It is the first time to construct recombinant adenovirus containing MOMP gene of avian chlamydia psittaci on the world.(1) Avian Chlamydia psittaci were isolated from chicken which were suspected suffering from Chlamydia psittaci through iodine staining, chicken embryo vaccination, sulfadiazine sodium test and electron microscopic observation. The virulence of hnq strain was measured in chicken embryo, which can reach 1.8× 10~(-7)/0.2ELD_(50), CpL strain can reach 5.6×10~(10)/0.2 ELD_(50). The CpL strain was chosen as research object. The PCR method for chicken chlamydiosis with good specificity and sensitivity was established.(2) The MOMP genes were cloned and sequenced. Comparisons of the MOMP amino acid sequence of CpL with other strains of avian chlamydia psittaci showed that there was 89.7%, 88.7%, 82.3%, and 94.4% of homology of MOMP of CpL with duck strain, turkey, pigeon, and parrot, respectively.(3) Using the way of gene ligation and recombination, avian chlamydia psittaci MOMP genes were inserted into the region between CMV promoter and SV40 Poly (A) of pShuttle-CMV vector to form a gene expression cassette. The positive pShuttle-CMV vector and skeleton vector of adenovirus were recombined in BJ5183 competent cells. Then positive recombinant adenovirus was picked out and named as Ad-MOMP. Sequence detection showed that the sequence of MOMP gene in recombinant adenovirus hadn't any mutation. Detecting the target genes and products expressed in HEK293 cells with an indirect immunofluorescent assay and PCR technique after transfection, it was proved that the recombinant adenovirus could express the target genes efficiently. Neutralization test showed that there was no cross response between EDS_(76) positive serum and the recombinant adenovirus in vitro. The stability test showed that the recombinant adenovirus with MOMP gene could stably passage in HEK293 cells.(4) Anti-MOMP antibodys of vaccinated SPF chicks were detected on 21~(th), the result showed that it can reach 1 : 32. SPF chicks vaccinated with 6.8×10~9TCID_(50) Ad-MOMP recombinant adenovirus could resist 5.6×10~(10)ELD_(50) CpL strain challenge on the day 21 post vaccination(dpv) and the protection rate was 9/10. But no chicks of both wild type adenovirus and HEK293 groups were protected from the challenge. No antibody against EDS_(76) was detected, which suggested that inoculation of Ad-MOMP recombinant adenovirus to chicks would not inhibit vaccination against
EDS76- Intramuscular and subcutaneous administrations were chosen in the test. The results suggested that the efficacy of both administrations was same. In order to meet practicability, we selected Intramuscular administration of the vaccine in the test.(5) According to the test results of SPF chicks, 7 days old chicks were vaccinated with 6.8 X lO9TCID5o Ad-MOMP recombinant adenovirus and challenged with the vaccine strain after 21 days. The results showed that chicks of the immunized group could resist 1.1 X 10I0ELD50 of the vaccine strain attacking while the control group failed after 6 months. The protection rate was 8/10. Antibody duration test showed that anti-MOMP antibodys can reach 1 : 64 on 21th day post vaccination. Then began down slowly still keeping a protective level on the 180th day. Specific antibody and T lymphocyte proliferation were detected with the IHA for Chlamydia antibody and MTT. The results showed that both of them had responses to Ad-MOMP recombinant adenovirus. The safety tests confirmed that the Ad-MOMP recombinant adenovirus had no side-effects on the inoculated chicks' health and gain and no recombinant adenovirus was discharged outside. In conservation period test, when the Ad-MOMP recombinant adenovirus was kept at -20°Cfor a year, its violence titer fell a little down, but immunity efficacy of the vaccine was un-reduced.
(1) 从疑似患鹦鹉热衣原体病鸡分离到衣原体,经涂片观察、接种鸡胚,碘染色试验、磺胺嘧啶钠试验、电子显微镜观察,确定为鹦鹉热衣原体,命名为hnq株,用鸡胚法测定其毒力为1.8×10~(-7)/0.2ELD_(50),CpL株的毒力为5.6×10~(10)/0.2 ELD_(50)。选择CpL株为研究对象。建立了鸡源鹦鹉热衣原体PCR诊断方法,并对其特异性、敏感性和灵敏度进行了验证。
(2) 对鸡源鹦鹉热衣原体CpL株MOMP基因进行了克隆和测序,并与禽源鹦鹉热衣原体其他菌株的MOMP基因进行了序列比较。结果表明,与禽源衣原体鸭(GD)株的MOMP氨基酸序列同源性为89.7%;与火鸡(CT1)株MOMP氨基酸序列同源性为88.7%;与鸽子(CP3)株MOMP氨基酸序列同源性为82.3%;与长尾小鹦鹉(VS225)株的MOMP氨基酸序列同源性为94.4%。
(3) 通过基因操作的方法,将鸡源鹦鹉热衣原体CpL株MOMP基因置于腺病毒穿梭载体的巨细胞瘤(CMV)启动子和SV40 Poly(A)尾巴之间,构建成目的基因的表达盒。构建的穿梭载体与腺病毒骨架载体在BJ5183工程菌内进行同源重组,筛选阳性重组腺病毒质粒,命名为Ad-MOMP。序列测定表明,Ad-MOMP重组腺病毒所带的鸡源鹦鹉热衣原体MOMP基因的氨基酸序列完全正确。转染HEK293细胞,用间接免疫荧光对表达产物检测,证明所构建的重组腺病毒能正确表达目的基因。减蛋综合征(EDS_(76))阳性血清中和试验表明,不能中和重组腺病毒。重组腺病毒稳定性试验表明,该重组病毒能稳定携带外源基因进行传代(23代)。
(4) 禽源鹦鹉热衣原体MOMP基因重组腺病毒免疫SPF小鸡,第21天测定抗MOMP抗体,达到1:32,用6.8×10~9TCID_(50)免疫SPF小鸡。能够抵抗5.6×10~10ELD_(50)同源强毒CpL株的攻击获得保护,保护率达9/10;用减蛋综合征病毒(EDS_(76)V)检测重组腺病毒抗体,表明二者无抗原—抗体交叉反应。用野生型腺病毒和HEK293细胞免疫的对照组均未产生任何的免疫保护效果。肌肉与皮下两种免疫途径试验结果表明,二者基本无差别,考虑到田间免疫的实用性,选择肌肉免疫。
(5)根据SPF小鸡免疫攻毒试验结果,用6.8×10~9TCID_(50)重组腺病毒一次肌肉免疫7日龄小鸡,21天后用同源强毒CpL株进行攻击,观察结果6个月,表明免疫小鸡能够抵抗1.1×10~10ELD_(50)鹦鹉热衣原体强毒的攻击,保护率达8/10。对照组10只鸡剖检后全部表现出鹦鹉热衣原体症状。抗体持续期试验表明,免疫后21天抗体水平达到最高(1:64),随后开始逐渐下降,至180天时,抗体水平仍然维持在保护水平。应用衣原体IHA和四甲基偶氮唑盐(MTT)法检测了小鸡的体液免疫和细胞免疫,表明二者都有应答。重组腺病毒安全性试验表明,小鸡生长正常,无排毒现象。重组腺病毒保存期试验显示,-20℃保存1年后 该重组腺病毒滴度有所下降,但不影响免疫力。
A chlamydia psittaci were isolated from affected chickens and the PCR method for chicken chlamydiosis was established. Recombinant adenovirus containing major outer membrane protein (MOMP) gene of Chlamydia psittaci of chicken origin were successfully constructed with human adenovirus type 5 vector which lack El and E3 genes. The MOMP genes were expressed in HEK293 cells and the recombinant adenovirus were evaluated in vitro and in vivo. It is the first time to construct recombinant adenovirus containing MOMP gene of avian chlamydia psittaci on the world.(1) Avian Chlamydia psittaci were isolated from chicken which were suspected suffering from Chlamydia psittaci through iodine staining, chicken embryo vaccination, sulfadiazine sodium test and electron microscopic observation. The virulence of hnq strain was measured in chicken embryo, which can reach 1.8× 10~(-7)/0.2ELD_(50), CpL strain can reach 5.6×10~(10)/0.2 ELD_(50). The CpL strain was chosen as research object. The PCR method for chicken chlamydiosis with good specificity and sensitivity was established.(2) The MOMP genes were cloned and sequenced. Comparisons of the MOMP amino acid sequence of CpL with other strains of avian chlamydia psittaci showed that there was 89.7%, 88.7%, 82.3%, and 94.4% of homology of MOMP of CpL with duck strain, turkey, pigeon, and parrot, respectively.(3) Using the way of gene ligation and recombination, avian chlamydia psittaci MOMP genes were inserted into the region between CMV promoter and SV40 Poly (A) of pShuttle-CMV vector to form a gene expression cassette. The positive pShuttle-CMV vector and skeleton vector of adenovirus were recombined in BJ5183 competent cells. Then positive recombinant adenovirus was picked out and named as Ad-MOMP. Sequence detection showed that the sequence of MOMP gene in recombinant adenovirus hadn't any mutation. Detecting the target genes and products expressed in HEK293 cells with an indirect immunofluorescent assay and PCR technique after transfection, it was proved that the recombinant adenovirus could express the target genes efficiently. Neutralization test showed that there was no cross response between EDS_(76) positive serum and the recombinant adenovirus in vitro. The stability test showed that the recombinant adenovirus with MOMP gene could stably passage in HEK293 cells.(4) Anti-MOMP antibodys of vaccinated SPF chicks were detected on 21~(th), the result showed that it can reach 1 : 32. SPF chicks vaccinated with 6.8×10~9TCID_(50) Ad-MOMP recombinant adenovirus could resist 5.6×10~(10)ELD_(50) CpL strain challenge on the day 21 post vaccination(dpv) and the protection rate was 9/10. But no chicks of both wild type adenovirus and HEK293 groups were protected from the challenge. No antibody against EDS_(76) was detected, which suggested that inoculation of Ad-MOMP recombinant adenovirus to chicks would not inhibit vaccination against
EDS76- Intramuscular and subcutaneous administrations were chosen in the test. The results suggested that the efficacy of both administrations was same. In order to meet practicability, we selected Intramuscular administration of the vaccine in the test.(5) According to the test results of SPF chicks, 7 days old chicks were vaccinated with 6.8 X lO9TCID5o Ad-MOMP recombinant adenovirus and challenged with the vaccine strain after 21 days. The results showed that chicks of the immunized group could resist 1.1 X 10I0ELD50 of the vaccine strain attacking while the control group failed after 6 months. The protection rate was 8/10. Antibody duration test showed that anti-MOMP antibodys can reach 1 : 64 on 21th day post vaccination. Then began down slowly still keeping a protective level on the 180th day. Specific antibody and T lymphocyte proliferation were detected with the IHA for Chlamydia antibody and MTT. The results showed that both of them had responses to Ad-MOMP recombinant adenovirus. The safety tests confirmed that the Ad-MOMP recombinant adenovirus had no side-effects on the inoculated chicks' health and gain and no recombinant adenovirus was discharged outside. In conservation period test, when the Ad-MOMP recombinant adenovirus was kept at -20°Cfor a year, its violence titer fell a little down, but immunity efficacy of the vaccine was un-reduced.
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