APP apxIVA基因序列特性及rApxIVA/rTbpB亚单位复合菌苗对小鼠的免疫研究
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摘要
猪传染性胸膜肺炎(Porcine Infectious Pleuropneumonia Syndrome,PIPS)是由胸膜肺炎放线杆菌(Actinobacillus Pleuropneumoniae,APP)引起的一种猪高度接触性呼吸道传染病,是猪急性呼吸道疾病发生的重要原因。由于引起PIPS的病原血清型众多,毒力因子复杂,不同血清型细菌毒力因子构成不同,使疫苗免疫难以达到令人满意的效果。
本研究以探讨APP两种在体内表达的蛋白ApxⅣA(胸膜肺炎放线杆菌RTX毒素ⅣA)和TbpB(运铁蛋白结合蛋白B)对灭活疫苗免疫效果的影响为目的。研究从分析基因序列特性开始,采用PCR方法成功扩增APP血清1型apxⅣA基因5'端两个特征性氨基酸序列(N端疏水性区域和中部乙酰化位点区域)的编码序列,构建这两段序列的表达载体apxⅣA1-pET32a和apxⅣA2-pET32a。将所构建表达载体和实验室前期构建并鉴定的tbpB-pET32a载体分别转化表达宿主菌大肠杆菌BL21,在0.6 mmol/L IPTG浓度条件下诱导3 h表达重组蛋白rApxⅣA1、rApxⅣA2和rTbpB。Western-blot分析表明rApxⅣA1和rTbpB具有良好的抗体反应原性,rApxⅣA2的抗体反应原性很弱。
rApxⅣA1和rTbpB作为亚单位疫苗按25μg/只/次的剂量分三次免疫小鼠,每次间隔两周,抗体监测结果表明两种重组蛋白单独或者是联合免疫小鼠都能诱导机体快速产生高水平抗体,在二免两周后抗体水平就达到活菌感染诱导机体产生的抗体水平。用APP1型4074株培养物制备灭活疫苗免疫小鼠(4×10~8CFU/只),免疫方法同重组蛋白。抗体监测结果表明灭活疫苗免疫小鼠能诱导机体产生ApxⅠ抗体、ApxⅡ抗体和LPS1抗体,不能诱导CPS1抗体产生。将重组蛋白与细菌培养物混合后制备亚单位复合菌苗免疫小鼠,抗体监测结果表明,灭活苗不会影响重组蛋白诱导机体产生抗体的能力;重组蛋白的添加,对灭活疫苗中部分抗原诱导机体产生抗体的能力有一定影响。在所检测抗体中,灭活疫苗诱导Apx1和LPS1抗体产生的能力受重组蛋白的影响比较明显,ApxⅡ抗体产生能力不受影响。三免后MTT法分析各免疫组小鼠脾细胞增殖能力,结果表明重组蛋白的添加量对脾细胞增殖能力有影响,重组蛋白50μg(rTbpB和rApxⅣA1各25μg)免疫组小鼠脾细胞增殖能力最强,重组蛋白25μg免疫组小鼠脾细胞增殖能力低于50μg免疫组;25μg免疫组中,不同重组蛋白免疫效果差异不明显:在所有免疫组中,灭活苗免疫组小鼠脾细胞增殖能力最弱。
攻毒试验结果表明,重组蛋白对同型(1型)细菌攻毒具有一定的免疫保护作用,rTbpB亚单位疫苗免疫能提供30%的保护率,rApxⅣA1亚单位疫苗免疫能提供20%的保护率,两者联合免疫能提供的免疫保护率为40%。灭活疫苗单独免疫提供的保护率为50%;与rTbpB或rApxⅣA联合免疫的保护率均为60%;与两种蛋白联合免疫提供的保护率为80%,重组蛋白能提高灭活疫苗的免疫保护率。对攻毒后死亡小鼠肺脏和存活一周的小鼠肺脏带菌量进行定量检测,结果表明不同疫苗免疫对APP的清除率不同。rTbpB免疫有助于降低细菌在肺脏的定殖能力,灭活疫苗免疫组小鼠肺脏带菌量在攻毒后一周变化不明显,rApxⅣA1免疫组小鼠肺脏带菌量在攻毒后一周出现明显上升。对于不同血清型(6型)攻毒,研究中所使用疫苗免疫后都不能产生很好的保护作用,除rApxⅣA/rTbpB亚单位复合菌苗免疫组有一只小鼠存活外,其余均在攻毒后死亡。
在对apxⅣA基因3'端片段的克隆研究中,发现该片段在基因组外复制时具有特殊性,即PCR扩增能形成不同大小的扩增片段,T克隆后重复序列缺失重复结构。序列分析表明,apxⅣA基因重复区域序列由4个重复单元组成,长度分别为159 bp、144 bp、135 bp和84 bp,在L20株、HV114株和4074株apxⅣA基因中的排列方式可以表示为[(P_(144bp))(P_(159bp))(P_(135bp))]m[(P_(144bp))(P_(159bp))(P_(84bp))(P_(144bp))(P_(159bp))(P_(135bp))]n(P_(144 bp))(m=0 or 1、n≥1)。在紧邻重复区域的序列中设计引物扩增该重复区域,电泳显示PCR扩增产物中有多种扩增条带。回收1型4074株扩增产物中约1800 bp、1200 bp和600 bp的三种片段,与pGMT连接后转化大肠杆菌,获得重组质粒pGMT-A、pGMT-B和pGMT-C。测序结果显示,三种质粒中的插入片段核酸序列为扩增区序列的一部分,具体为上游引物结合位点到重复区之间39 bp、下游引物结合位点到重复区36 bp和144 bp重复单元在3'末端的一个重复。T克隆结果表明,PCR扩增产物中三种不同大小的产物都是扩增区域PCR扩增的结果,即同一模板经PCR扩增产生了不同大小的产物;这些产物插入T载体随质粒在大肠杆菌中复制后,丢失全部重复结构,仅留重复区1794 bp中的144 bp与重复区两侧序列重新连接成一个无重复结构的普通序列。对apxⅣA基因重复序列在基因组外复制稳定性的研究表明,重复序列在复制过程中具有丢失部分重复单元并最终丢失整个重复结构变成非重复序列的特性。这一特性与ApxⅣA蛋白的自我剪切活性是否有因果联系,以及这一现象是否在细菌基因组复制过程中同样发生,还有待进一步试验研究。
Actinobacillus pleuropneumoniae(APP) is the causative agent of porcine infectious pleuropneumonia syndrome(PIPS),a fibrinous,exudative,hemorrhagic,necrotizing pleuropneumonia affecting all ages of pigs.The virulence of A.pleuropneumoniae is known to be variable.BiotypeⅠhas been divided into 13 serotypes and biotypeⅡinto 2 serotypes, for a total of 15 serotypes.And virulence factors to compose the virulence of A.pleuropneumoniae are complex.Furthermore,strains of different serotype contain different virulence factors.Though widely used,inactive vaccine could not induce mice to absolutely resist attack of APP for reasons mentioned above.
The object of this study is to find the effects of two proteins,transferin binding protein B (TbpB) and actinobacillus pleuropneumoniae RTX toxin IVA(ApxIVA) on the protection ability of inactive vaccine in mice.At first part of the study,the characteristics of apxIVA was analysed by bioinformatics method to find the conserved region and variable region.For the variable region,repeat sequence analysis was conducted to confirm the region of repeat region,the repeat units and the arrangement of them.For the conserved region,fragments encoding two characteristic amino acid sequence,N end hydrophobic region and middle part potential acylation sites of ApxIVA,were PCR amplified to construct expression plasmids apxIVA1-pET32a and apxIVA1-pET32a.Another expression plasmid tbpB-pET32 constructed in previous research and expression plasmids constructed in this study were transformed into expression host cell E.coli BL21 to express rApxIVA1,rApxIVA2 and rTbpB with the induction of 0.6 mmol/L IPTG for 3 hours.Results of western-blot analysis showed that rApxIVA1 and rTbpB could strongly bind antibody react with positive sera prepared by inoculating mice with live bacteria.On the other hand,rApxIVA2 only possesses weak antibody reaction ability.
25μg of rApxIVA1 and 25μg of rTbpB were used as subunit vaccines to immunize mice for three times with interval of two weeks by intraperitoneal injection.Results of antibody detection indicated that two recombinant proteins could induce mice to produce high level of antibodies against them after two times of immunization.Inactive vaccine prepared by inactivating the culture of strain 4074 by formalin was used to immunize mice with dosage of 1×10~9CFU per mouse using the same method as used for subunit vaccines.Results of antibody detection indicated that inactive vaccine could induce mice to produce antibodies against ApxⅠ,ApxⅡand LPS1.The level of antibodies against CPS1 was not significant with those in negative control.The level of antibodies against ApxⅠ,ApxⅡand LPS1 were all significantly lower than those produced by infection of live bacteria(P＜0.01).When the mice were vaccinated with vaccines prepared by mixing recombinant proteins with formalin-inactivated bacteria,the level of antibodies against ApxⅠand LPS1 were significantly lower than those induced by vaccination with single inactive vaccine.But the antibodies against recombinant proteins were not affected by the addition of bacteria.
One week after the third immune,spleens of three mice in each group were collected for proliferation activity test.Results of MTT assay demonstrated that the proliferation activity of spleen cells from mice vaccinated with 50μg recombinant protein(25μg of rTbpB and 25μg rApxIVA1) is significantly stronger than other groups.The proliferation activity of spleen cells from mice vaccinated with inactive vaccine is significantly weaker than other groups.
Part of the mice,30%in group vaccinated with rTbpB,20%in group immunized by rApxIVA1,40%in group vaccinated with the mixture of both rTbpB and rApxIVA1 survived from the challenge of strain 4074,serotypel,from which the nucleotides for the construction of tbpB-pET and apxIVA1-pET derived.Compared to single inactive vaccine,the protection rate against challenge of strain 4074 was raised by immunization with the mixture of inactive vaccine and either rTbpB,rApxIVA1 or both.After challenge of strain 4074,only 50%of mice survived in groups vaccinated with single inactive vaccine.When combined with rThpB or rApxIVA1,the protection rates were improved to 60%.Further more,80%mice in group vaccinated with the mixture of inactive vaccine and both recombinant proteins.
The number of APP either in lungs of mice died right after challenge or survived for one week from challenge day were detected by Real time PCR method.Compared to the number of APP in lungs of dead mice,there was significant difference among groups vaccinated with different vaccines.Vaccination with rTbpB helps to decrease the number of APP in lungs.No significant change is detected in groups vaccinated with inactivate vaccine.The number of APP existed in lungs was raised greatly,almost ten times of those detected in dead mice.
After challenge with strain Fem,serotype 6,almost all the mice tested died,with the exception of only mouse in group vaccinated with the mixture of inactive vaccine and two recombinant proteins.The results in challenge test demonstrated that,when used as vaccine, two recombinant proteins could provide some degree of protection only to the challenge of same strain.
The significant characteristic,which was discovered during the course of clone,of apxIVA gene is that repeat units in repeat region would lose and finally change into non-repeated sequence when they duplicate outside genome of APP.Sequence analysis shows that apxIVA gene consists of 4 kind of repeated units(with length of 159bp,144 bp,135 bp and 84 bp,respectively).The arrangement of repeat units in apxIVA gene of strain L20,strain HV114 and strain 4074 can be shown as[(P144 bp)(P159 bp)(P135 bp)]m[(P144 bp)(P159 bp)(P84 bp)(P144 bp)(P159 bp)(P135 bp)]n(P144 bp)(m=0 or 1、n≥1).Primers were selected from sequences just adjacent the repeat structure to amplify this repeated area.Gel electrophoretic analysis of PCR products showed there were more than one bands.Bands with size of about 1800 bp,1200 bp,600 bp from the PCR products amplified from genome of 4074 were reclaimed and transformed into E coli after being ligated with pGMT vector. Sequencings results demonstrated that obtained plasmids from the clones of different size of fragments contained completely same inserted nucleotide sequences,which was part of amplification area sequence,39 bp from the binding site of upstream primer to repeat region, 36 bp from the binding site of lower primer to the 3end of repeat region,the other 144 bp from one repeat of 144 bp repeat unit located at 3end part of repeat region.T clone results indicated that PCR amplification from the same template gave different size of products.And they would get into same sequence with the loss of all repeated sequence after replication in plasmid.Whether this phenomenon has causal association with the self-processing "clip and link" activity observed by Osicka,or whether the same phenomenon occurs in bacterial genome replication need further study to discover.
本研究以探讨APP两种在体内表达的蛋白ApxⅣA(胸膜肺炎放线杆菌RTX毒素ⅣA)和TbpB(运铁蛋白结合蛋白B)对灭活疫苗免疫效果的影响为目的。研究从分析基因序列特性开始,采用PCR方法成功扩增APP血清1型apxⅣA基因5'端两个特征性氨基酸序列(N端疏水性区域和中部乙酰化位点区域)的编码序列,构建这两段序列的表达载体apxⅣA1-pET32a和apxⅣA2-pET32a。将所构建表达载体和实验室前期构建并鉴定的tbpB-pET32a载体分别转化表达宿主菌大肠杆菌BL21,在0.6 mmol/L IPTG浓度条件下诱导3 h表达重组蛋白rApxⅣA1、rApxⅣA2和rTbpB。Western-blot分析表明rApxⅣA1和rTbpB具有良好的抗体反应原性,rApxⅣA2的抗体反应原性很弱。
rApxⅣA1和rTbpB作为亚单位疫苗按25μg/只/次的剂量分三次免疫小鼠,每次间隔两周,抗体监测结果表明两种重组蛋白单独或者是联合免疫小鼠都能诱导机体快速产生高水平抗体,在二免两周后抗体水平就达到活菌感染诱导机体产生的抗体水平。用APP1型4074株培养物制备灭活疫苗免疫小鼠(4×10~8CFU/只),免疫方法同重组蛋白。抗体监测结果表明灭活疫苗免疫小鼠能诱导机体产生ApxⅠ抗体、ApxⅡ抗体和LPS1抗体,不能诱导CPS1抗体产生。将重组蛋白与细菌培养物混合后制备亚单位复合菌苗免疫小鼠,抗体监测结果表明,灭活苗不会影响重组蛋白诱导机体产生抗体的能力;重组蛋白的添加,对灭活疫苗中部分抗原诱导机体产生抗体的能力有一定影响。在所检测抗体中,灭活疫苗诱导Apx1和LPS1抗体产生的能力受重组蛋白的影响比较明显,ApxⅡ抗体产生能力不受影响。三免后MTT法分析各免疫组小鼠脾细胞增殖能力,结果表明重组蛋白的添加量对脾细胞增殖能力有影响,重组蛋白50μg(rTbpB和rApxⅣA1各25μg)免疫组小鼠脾细胞增殖能力最强,重组蛋白25μg免疫组小鼠脾细胞增殖能力低于50μg免疫组;25μg免疫组中,不同重组蛋白免疫效果差异不明显:在所有免疫组中,灭活苗免疫组小鼠脾细胞增殖能力最弱。
攻毒试验结果表明,重组蛋白对同型(1型)细菌攻毒具有一定的免疫保护作用,rTbpB亚单位疫苗免疫能提供30%的保护率,rApxⅣA1亚单位疫苗免疫能提供20%的保护率,两者联合免疫能提供的免疫保护率为40%。灭活疫苗单独免疫提供的保护率为50%;与rTbpB或rApxⅣA联合免疫的保护率均为60%;与两种蛋白联合免疫提供的保护率为80%,重组蛋白能提高灭活疫苗的免疫保护率。对攻毒后死亡小鼠肺脏和存活一周的小鼠肺脏带菌量进行定量检测,结果表明不同疫苗免疫对APP的清除率不同。rTbpB免疫有助于降低细菌在肺脏的定殖能力,灭活疫苗免疫组小鼠肺脏带菌量在攻毒后一周变化不明显,rApxⅣA1免疫组小鼠肺脏带菌量在攻毒后一周出现明显上升。对于不同血清型(6型)攻毒,研究中所使用疫苗免疫后都不能产生很好的保护作用,除rApxⅣA/rTbpB亚单位复合菌苗免疫组有一只小鼠存活外,其余均在攻毒后死亡。
在对apxⅣA基因3'端片段的克隆研究中,发现该片段在基因组外复制时具有特殊性,即PCR扩增能形成不同大小的扩增片段,T克隆后重复序列缺失重复结构。序列分析表明,apxⅣA基因重复区域序列由4个重复单元组成,长度分别为159 bp、144 bp、135 bp和84 bp,在L20株、HV114株和4074株apxⅣA基因中的排列方式可以表示为[(P_(144bp))(P_(159bp))(P_(135bp))]m[(P_(144bp))(P_(159bp))(P_(84bp))(P_(144bp))(P_(159bp))(P_(135bp))]n(P_(144 bp))(m=0 or 1、n≥1)。在紧邻重复区域的序列中设计引物扩增该重复区域,电泳显示PCR扩增产物中有多种扩增条带。回收1型4074株扩增产物中约1800 bp、1200 bp和600 bp的三种片段,与pGMT连接后转化大肠杆菌,获得重组质粒pGMT-A、pGMT-B和pGMT-C。测序结果显示,三种质粒中的插入片段核酸序列为扩增区序列的一部分,具体为上游引物结合位点到重复区之间39 bp、下游引物结合位点到重复区36 bp和144 bp重复单元在3'末端的一个重复。T克隆结果表明,PCR扩增产物中三种不同大小的产物都是扩增区域PCR扩增的结果,即同一模板经PCR扩增产生了不同大小的产物;这些产物插入T载体随质粒在大肠杆菌中复制后,丢失全部重复结构,仅留重复区1794 bp中的144 bp与重复区两侧序列重新连接成一个无重复结构的普通序列。对apxⅣA基因重复序列在基因组外复制稳定性的研究表明,重复序列在复制过程中具有丢失部分重复单元并最终丢失整个重复结构变成非重复序列的特性。这一特性与ApxⅣA蛋白的自我剪切活性是否有因果联系,以及这一现象是否在细菌基因组复制过程中同样发生,还有待进一步试验研究。
Actinobacillus pleuropneumoniae(APP) is the causative agent of porcine infectious pleuropneumonia syndrome(PIPS),a fibrinous,exudative,hemorrhagic,necrotizing pleuropneumonia affecting all ages of pigs.The virulence of A.pleuropneumoniae is known to be variable.BiotypeⅠhas been divided into 13 serotypes and biotypeⅡinto 2 serotypes, for a total of 15 serotypes.And virulence factors to compose the virulence of A.pleuropneumoniae are complex.Furthermore,strains of different serotype contain different virulence factors.Though widely used,inactive vaccine could not induce mice to absolutely resist attack of APP for reasons mentioned above.
The object of this study is to find the effects of two proteins,transferin binding protein B (TbpB) and actinobacillus pleuropneumoniae RTX toxin IVA(ApxIVA) on the protection ability of inactive vaccine in mice.At first part of the study,the characteristics of apxIVA was analysed by bioinformatics method to find the conserved region and variable region.For the variable region,repeat sequence analysis was conducted to confirm the region of repeat region,the repeat units and the arrangement of them.For the conserved region,fragments encoding two characteristic amino acid sequence,N end hydrophobic region and middle part potential acylation sites of ApxIVA,were PCR amplified to construct expression plasmids apxIVA1-pET32a and apxIVA1-pET32a.Another expression plasmid tbpB-pET32 constructed in previous research and expression plasmids constructed in this study were transformed into expression host cell E.coli BL21 to express rApxIVA1,rApxIVA2 and rTbpB with the induction of 0.6 mmol/L IPTG for 3 hours.Results of western-blot analysis showed that rApxIVA1 and rTbpB could strongly bind antibody react with positive sera prepared by inoculating mice with live bacteria.On the other hand,rApxIVA2 only possesses weak antibody reaction ability.
25μg of rApxIVA1 and 25μg of rTbpB were used as subunit vaccines to immunize mice for three times with interval of two weeks by intraperitoneal injection.Results of antibody detection indicated that two recombinant proteins could induce mice to produce high level of antibodies against them after two times of immunization.Inactive vaccine prepared by inactivating the culture of strain 4074 by formalin was used to immunize mice with dosage of 1×10~9CFU per mouse using the same method as used for subunit vaccines.Results of antibody detection indicated that inactive vaccine could induce mice to produce antibodies against ApxⅠ,ApxⅡand LPS1.The level of antibodies against CPS1 was not significant with those in negative control.The level of antibodies against ApxⅠ,ApxⅡand LPS1 were all significantly lower than those produced by infection of live bacteria(P＜0.01).When the mice were vaccinated with vaccines prepared by mixing recombinant proteins with formalin-inactivated bacteria,the level of antibodies against ApxⅠand LPS1 were significantly lower than those induced by vaccination with single inactive vaccine.But the antibodies against recombinant proteins were not affected by the addition of bacteria.
One week after the third immune,spleens of three mice in each group were collected for proliferation activity test.Results of MTT assay demonstrated that the proliferation activity of spleen cells from mice vaccinated with 50μg recombinant protein(25μg of rTbpB and 25μg rApxIVA1) is significantly stronger than other groups.The proliferation activity of spleen cells from mice vaccinated with inactive vaccine is significantly weaker than other groups.
Part of the mice,30%in group vaccinated with rTbpB,20%in group immunized by rApxIVA1,40%in group vaccinated with the mixture of both rTbpB and rApxIVA1 survived from the challenge of strain 4074,serotypel,from which the nucleotides for the construction of tbpB-pET and apxIVA1-pET derived.Compared to single inactive vaccine,the protection rate against challenge of strain 4074 was raised by immunization with the mixture of inactive vaccine and either rTbpB,rApxIVA1 or both.After challenge of strain 4074,only 50%of mice survived in groups vaccinated with single inactive vaccine.When combined with rThpB or rApxIVA1,the protection rates were improved to 60%.Further more,80%mice in group vaccinated with the mixture of inactive vaccine and both recombinant proteins.
The number of APP either in lungs of mice died right after challenge or survived for one week from challenge day were detected by Real time PCR method.Compared to the number of APP in lungs of dead mice,there was significant difference among groups vaccinated with different vaccines.Vaccination with rTbpB helps to decrease the number of APP in lungs.No significant change is detected in groups vaccinated with inactivate vaccine.The number of APP existed in lungs was raised greatly,almost ten times of those detected in dead mice.
After challenge with strain Fem,serotype 6,almost all the mice tested died,with the exception of only mouse in group vaccinated with the mixture of inactive vaccine and two recombinant proteins.The results in challenge test demonstrated that,when used as vaccine, two recombinant proteins could provide some degree of protection only to the challenge of same strain.
The significant characteristic,which was discovered during the course of clone,of apxIVA gene is that repeat units in repeat region would lose and finally change into non-repeated sequence when they duplicate outside genome of APP.Sequence analysis shows that apxIVA gene consists of 4 kind of repeated units(with length of 159bp,144 bp,135 bp and 84 bp,respectively).The arrangement of repeat units in apxIVA gene of strain L20,strain HV114 and strain 4074 can be shown as[(P144 bp)(P159 bp)(P135 bp)]m[(P144 bp)(P159 bp)(P84 bp)(P144 bp)(P159 bp)(P135 bp)]n(P144 bp)(m=0 or 1、n≥1).Primers were selected from sequences just adjacent the repeat structure to amplify this repeated area.Gel electrophoretic analysis of PCR products showed there were more than one bands.Bands with size of about 1800 bp,1200 bp,600 bp from the PCR products amplified from genome of 4074 were reclaimed and transformed into E coli after being ligated with pGMT vector. Sequencings results demonstrated that obtained plasmids from the clones of different size of fragments contained completely same inserted nucleotide sequences,which was part of amplification area sequence,39 bp from the binding site of upstream primer to repeat region, 36 bp from the binding site of lower primer to the 3end of repeat region,the other 144 bp from one repeat of 144 bp repeat unit located at 3end part of repeat region.T clone results indicated that PCR amplification from the same template gave different size of products.And they would get into same sequence with the loss of all repeated sequence after replication in plasmid.Whether this phenomenon has causal association with the self-processing "clip and link" activity observed by Osicka,or whether the same phenomenon occurs in bacterial genome replication need further study to discover.
引文
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