抗日本血吸虫生殖产卵编码基因pVAX1/SjHGPRT·SDISP多价疫苗的构建及功能的研究
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摘要
本实验室前期的研究已经证实,日本血吸虫未成熟虫卵可溶性抗原26-28kDa(SIEA26-28kDa)天然分子可诱导宿主产生较好的抗雌虫生殖产卵、抗卵胚发育的免疫效果,但由于在批量生产中存在着难以克服的瓶颈,不可避免地制约了其在实际中的应用。因此,我们通过SIEA26-28kDa抗血清免疫识别、肽指纹图谱、质谱分析,获得了同源性较高、免疫原性较强的蛋白质斑点,其中包括与日本血吸虫次黄嘌呤。鸟嘌呤磷酸核糖转移酶(HGPRT),琥珀酸脱氢酶铁硫蛋白(SDISP)相匹配的斑点,并证实HGPRT、SDISP分别与细胞的核苷酸代谢、血吸虫的生长发育、生理代谢密切相关,可以作为抗日本血吸虫病天然分子基因疫苗研究的靶标。在此基础上本实验室克隆了SjHGPRT和SjSDISP全长cDNA,并以SjHGPRT和SjSDISP基因为目的片段构建了单价核酸疫苗pcDNA3.0/SjHGPRT和pcDNA3.0/SjSDISP,其动物实验及安全性评价显示该核酸疫苗具有较好的免疫保护力,且对家兔局部肌肉刺激反应轻微,对豚鼠无全身过敏反应。但单一的核酸疫苗在体内诱导出的有效的保护力尚不足以完全。为了能进一步提高抗日本血吸虫生殖产卵编码相关基因SjHGPRT和SjSDISP单价核酸疫苗的保护效果,本研究充分利用DNA疫苗易于构建,易于生产的特点,将SjHGPRT、SjSDISP的编码基因以“串联”方式插入到同一人用载体pVAX1上,构建多价共表达DNA疫苗pVAX1/SjHGPRT·SDISP,并进行动物实验,观察其免疫保护力。
研究目的
1、针对抗日本血吸虫生殖产卵编码基因SjHGPRT、SjSDISP,构建单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP及多价疫苗pVAX1/SjHGPRT·SDISP,并获得良好的表达;
2、观察并比较单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP和多价疫苗pVAX1/SjHGPRT·SDISP免疫小鼠产生的保护力。
研究方法
1、单价疫苗pVAX1/SjGPRT、pVAX1/SjSDISP及多价疫苗pVAX1/SjHGPRT·SDISP的构建。
分别以pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP为模板,设计引物扩增SjHGPRT、SjSDISP,用overlap法扩增全长的SjHGPRT·SDISP,分别将扩增产物鉴定后与真核质粒载体pVAX1连接,转化至DH5α细胞。对三种阳性克隆鉴定并测序证实之。
2、单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP和多价疫苗SjHGPRT·SDISP的表达,及其对免疫小鼠的保护性效果观察。
10只昆明小鼠随机分成5组:A组为pVAX1/SjHGPRT·SDISP多价疫苗组,B组为pVAX1/SjSDISP免疫组,C组为pVAX1/SjHGPRT免疫组,D组为pVAX1对照组,E组为NS组。分别提取以上质粒,以100μg/只经左腿股四头肌注射昆明小鼠,14d后取肌肉组织切片进行酶免疫组织化学染色,同时设pVAX1空载体、NS阴性对照组,观察其表达。
50只昆明小鼠随机分成5组(A-D组同上),大量提取以上质粒,每只小鼠100gg或等量NS经左腿股四头肌注射进行免疫。4周后以20±1条尾蚴贴腹感染,感染6周后检测减虫率、减卵率。
研究结果
1、PCR、Kpn1和XbaⅠ双酶切及测序均显示重组质粒的插入序列分别与目的基因ORF完全一致,说明单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP和多价疫苗pVAX1/SjHGPRT·SDISP构建成功。
2、昆明小鼠肌肉组织酶免疫组织化学染色显示免疫组小鼠肌细胞中呈现较强的棕黄色颗粒,而阴性对照组肌细胞呈阴性。证实pVAX1/SjHGPRT、pVAX1/SjSDISP、pVAX1/SjHGPRT·SDISP均能在小鼠肌肉细胞内成功表达。
3、pVAX1/SjHGPRT·SDISP、pVAX1/SjSDISP、pVAX1/SjHGPRT免疫小鼠后分别获得了42.20%、36.40%、25.30%的减虫率,68.04%、23.41%、52.55%的每雌子宫减卵率和72.96%、45.40%、69.91%的每克肝减卵率,与NS组比较各组均有显著性差异(P＜0.05);pVAX1/SjHGPRT·SDISP、pVAX1/SjSDISP、pVAX1/SjHGPRT各免疫组与相应的空质粒组相比各指标均具有显著性差异(P＜0.05);多价疫苗pVAX1/SjHGPRT·SDISP组与单价疫苗组相比,减虫率、肝减卵率无显著性差异(P＞0.05),但每雌子宫减卵率有显著性差异(P＜0.05);单价疫苗pVAX1/SjHGPRT组与pVAX1/SjSDISP组相比,减虫率无显著性差异(P＞0.05),但肝减卵率和每雌子宫减卵率均有显著性差异(P＜0.01);单价与多价疫苗组的减卵率均明显高于减虫率。
结论
1、成功构建真核重组质粒pVAX1/SjHGPRT、pVAX1/SjSDISP、pVAX1/SjHGPRT·SDISP,并在昆明鼠肌肉细胞内均获得良好表达。
2、单价疫苗pVAX1/SjSDISP、pVAX1/SjHGPRT与多价疫苗pVAX1/SjHGPRT·SDISP免疫小鼠均能产生较好的保护力;多价疫苗pVAX1/SjHGPRT·SDISP组与单价疫苗组免疫小鼠产生相似的抗日本血吸虫攻击感染的保护力,但多价疫苗组免疫昆明小鼠的每雌子宫减卵率可以产生比单价疫苗更优的免疫保护效果;单价疫苗pVAX1/SjHGPRT组免疫小鼠后每雌子宫减卵率和肝减卵率效果明显优于pVAX1/sJSDISP组;构建的SjHGPRT和SjSDISP单价与多价疫苗,在抗雌虫减卵、抗卵胚发育和生殖的效果明显优于减虫效果。
It is proved that the soluble immature egg antigens(SIEA26-28kDa) from Schistosoma japonicum play an important role in anti-embryonation and anti-fecundity immunity.Later,we obtained three highly homologous and immunogenic proteins pots through immunological recognition with anti-sera against SIEA26-28kDa and methods of the peptide mass fingerprintmaps and MALDI-TOF-MS analysis.It include the protein spots relevanted to hypoxanthine-guanine phosphoribosyl transferase(HGPRT),succinate dehydrogenase iron-sulfur protein(SDISP).On this base several DNA recombinant vaccines related to the natural molecular SIEA26-28kDa,such as pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP,were constructed which have induced partial protection and be proved that it hardly had the risk of allergization to the host.But monovalent vaccine's protective efficacy needed to be further improved.So we constructed the multivalent DNA vaccine pVAX1/SjHGPRToSDISP and studied their immunoprotection effect.
Objective
1.Construct the monovalent vaccine pVAX1/SjHGPRT、pVAX1/ SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP which is the target gene against Schistosoma japonicum.
2.Study immunoprotection efficacy of the multivalent vaccine pVAX1/SjHGPRT·SDISP compared with the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP which induced in mice.
Methods
1.Construction of the monovalent vaccine pVAX1/SjHGPRT、pVAX 1/SjSDI SP and the multivalent vaccine pVAX1/SjHGPRT·SDISP
SjHGPRT、SjSDISP were amplified with the templates of pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP and spliced to SjHGPRT·SDISP by SOE- PCR(splicing by overlap extension),then the SjHGPRT,SjSDISP and SjHGPRToSDISP were cloned into the vector pVAX1 and transformed to the E.coli DH5αcells.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjHGPRT·SDISP were successfully constructed.
2.Expression and immunoprotection of the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP
Sixty mouse are divided to five group in radom.A: pVAX1/SjHGPRT·SDISP,B:pVAX1/SjSDISP,C:pVAX 1/SjHGPRT, D:pVAX1,E:NS.Extracting those plasmids and injecting the plasmids to quadriceps femoris of ten Kunming mise to confirm their successful expression by immunohistochemistry assay.Negative control,the plasmid pVAX1 and NS.Then we largely extracted those plasmids to fifty immune Kunming mise in five groups which followed by being challenged with cercariae of Schistosomajaponicum.Immunoprotection against Schistosoma japonicum in mice was evaluated by reduction of worm burden,intrauterine eggs and liver eggs.
Result
1.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjHGPRT, pVAX1/SjSDISP and pVAX1/SjHGPRToSDISP were successfully constructed.
2.Result of immunohistochemistry showed intense brown particles appeared in the muscle cells of Kunming mise immuned with the multivalent vaccine,while no specific fluorescence or brown particles appeared in muscle of the control groups.These results demonstrated the expression of the recombinant plasmids pVAX1/SjHGPRT, pVAX1/SjSDISP and pVAX1/SjHGPRTo SDISP were successful.
3.Compared with the NS group and pVAX1 group,both pVAX1/SjHGPRT·SDISP and monovalent vaccine pVAX1/SjSDISP、pVAX1/SjHGPRT group gained statistically significant protection result. Also statistically significant was induced compared to the monovalent vaccine group on reduction rate in uterus.In the same time statistically significant was attained between pVAX1/SjSDISP group and pVAX1/ SjHGPRT group.While the worm and the liver egg reduction rates between pVAX1/SjHGPRT·SDISP and monovalent vaccine group were no statistically significant.Among all the groups the egg reduction rates are higher than the worm reduction rates.
Conclusion
1.Both the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP are successfully constructed.Both the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP are successfully expressed in the quadriceps femoris of Kunming mise.
2.Both the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP can induced partial protection in mice.The multivalent vaccine pVAX1/SjHGPRT·SDISP can induce significant immunoprotection which is better than the monovalent vaccine on reduction rate in uterus.
研究目的
1、针对抗日本血吸虫生殖产卵编码基因SjHGPRT、SjSDISP,构建单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP及多价疫苗pVAX1/SjHGPRT·SDISP,并获得良好的表达;
2、观察并比较单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP和多价疫苗pVAX1/SjHGPRT·SDISP免疫小鼠产生的保护力。
研究方法
1、单价疫苗pVAX1/SjGPRT、pVAX1/SjSDISP及多价疫苗pVAX1/SjHGPRT·SDISP的构建。
分别以pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP为模板,设计引物扩增SjHGPRT、SjSDISP,用overlap法扩增全长的SjHGPRT·SDISP,分别将扩增产物鉴定后与真核质粒载体pVAX1连接,转化至DH5α细胞。对三种阳性克隆鉴定并测序证实之。
2、单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP和多价疫苗SjHGPRT·SDISP的表达,及其对免疫小鼠的保护性效果观察。
10只昆明小鼠随机分成5组:A组为pVAX1/SjHGPRT·SDISP多价疫苗组,B组为pVAX1/SjSDISP免疫组,C组为pVAX1/SjHGPRT免疫组,D组为pVAX1对照组,E组为NS组。分别提取以上质粒,以100μg/只经左腿股四头肌注射昆明小鼠,14d后取肌肉组织切片进行酶免疫组织化学染色,同时设pVAX1空载体、NS阴性对照组,观察其表达。
50只昆明小鼠随机分成5组(A-D组同上),大量提取以上质粒,每只小鼠100gg或等量NS经左腿股四头肌注射进行免疫。4周后以20±1条尾蚴贴腹感染,感染6周后检测减虫率、减卵率。
研究结果
1、PCR、Kpn1和XbaⅠ双酶切及测序均显示重组质粒的插入序列分别与目的基因ORF完全一致,说明单价疫苗pVAX1/SjHGPRT、pVAX1/SjSDISP和多价疫苗pVAX1/SjHGPRT·SDISP构建成功。
2、昆明小鼠肌肉组织酶免疫组织化学染色显示免疫组小鼠肌细胞中呈现较强的棕黄色颗粒,而阴性对照组肌细胞呈阴性。证实pVAX1/SjHGPRT、pVAX1/SjSDISP、pVAX1/SjHGPRT·SDISP均能在小鼠肌肉细胞内成功表达。
3、pVAX1/SjHGPRT·SDISP、pVAX1/SjSDISP、pVAX1/SjHGPRT免疫小鼠后分别获得了42.20%、36.40%、25.30%的减虫率,68.04%、23.41%、52.55%的每雌子宫减卵率和72.96%、45.40%、69.91%的每克肝减卵率,与NS组比较各组均有显著性差异(P＜0.05);pVAX1/SjHGPRT·SDISP、pVAX1/SjSDISP、pVAX1/SjHGPRT各免疫组与相应的空质粒组相比各指标均具有显著性差异(P＜0.05);多价疫苗pVAX1/SjHGPRT·SDISP组与单价疫苗组相比,减虫率、肝减卵率无显著性差异(P＞0.05),但每雌子宫减卵率有显著性差异(P＜0.05);单价疫苗pVAX1/SjHGPRT组与pVAX1/SjSDISP组相比,减虫率无显著性差异(P＞0.05),但肝减卵率和每雌子宫减卵率均有显著性差异(P＜0.01);单价与多价疫苗组的减卵率均明显高于减虫率。
结论
1、成功构建真核重组质粒pVAX1/SjHGPRT、pVAX1/SjSDISP、pVAX1/SjHGPRT·SDISP,并在昆明鼠肌肉细胞内均获得良好表达。
2、单价疫苗pVAX1/SjSDISP、pVAX1/SjHGPRT与多价疫苗pVAX1/SjHGPRT·SDISP免疫小鼠均能产生较好的保护力;多价疫苗pVAX1/SjHGPRT·SDISP组与单价疫苗组免疫小鼠产生相似的抗日本血吸虫攻击感染的保护力,但多价疫苗组免疫昆明小鼠的每雌子宫减卵率可以产生比单价疫苗更优的免疫保护效果;单价疫苗pVAX1/SjHGPRT组免疫小鼠后每雌子宫减卵率和肝减卵率效果明显优于pVAX1/sJSDISP组;构建的SjHGPRT和SjSDISP单价与多价疫苗,在抗雌虫减卵、抗卵胚发育和生殖的效果明显优于减虫效果。
It is proved that the soluble immature egg antigens(SIEA26-28kDa) from Schistosoma japonicum play an important role in anti-embryonation and anti-fecundity immunity.Later,we obtained three highly homologous and immunogenic proteins pots through immunological recognition with anti-sera against SIEA26-28kDa and methods of the peptide mass fingerprintmaps and MALDI-TOF-MS analysis.It include the protein spots relevanted to hypoxanthine-guanine phosphoribosyl transferase(HGPRT),succinate dehydrogenase iron-sulfur protein(SDISP).On this base several DNA recombinant vaccines related to the natural molecular SIEA26-28kDa,such as pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP,were constructed which have induced partial protection and be proved that it hardly had the risk of allergization to the host.But monovalent vaccine's protective efficacy needed to be further improved.So we constructed the multivalent DNA vaccine pVAX1/SjHGPRToSDISP and studied their immunoprotection effect.
Objective
1.Construct the monovalent vaccine pVAX1/SjHGPRT、pVAX1/ SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP which is the target gene against Schistosoma japonicum.
2.Study immunoprotection efficacy of the multivalent vaccine pVAX1/SjHGPRT·SDISP compared with the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP which induced in mice.
Methods
1.Construction of the monovalent vaccine pVAX1/SjHGPRT、pVAX 1/SjSDI SP and the multivalent vaccine pVAX1/SjHGPRT·SDISP
SjHGPRT、SjSDISP were amplified with the templates of pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP and spliced to SjHGPRT·SDISP by SOE- PCR(splicing by overlap extension),then the SjHGPRT,SjSDISP and SjHGPRToSDISP were cloned into the vector pVAX1 and transformed to the E.coli DH5αcells.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjHGPRT·SDISP were successfully constructed.
2.Expression and immunoprotection of the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP
Sixty mouse are divided to five group in radom.A: pVAX1/SjHGPRT·SDISP,B:pVAX1/SjSDISP,C:pVAX 1/SjHGPRT, D:pVAX1,E:NS.Extracting those plasmids and injecting the plasmids to quadriceps femoris of ten Kunming mise to confirm their successful expression by immunohistochemistry assay.Negative control,the plasmid pVAX1 and NS.Then we largely extracted those plasmids to fifty immune Kunming mise in five groups which followed by being challenged with cercariae of Schistosomajaponicum.Immunoprotection against Schistosoma japonicum in mice was evaluated by reduction of worm burden,intrauterine eggs and liver eggs.
Result
1.Restriction enzyme digestion,PCR and sequencing results confirmed that recombinant expression plasmids pVAX1/SjHGPRT, pVAX1/SjSDISP and pVAX1/SjHGPRToSDISP were successfully constructed.
2.Result of immunohistochemistry showed intense brown particles appeared in the muscle cells of Kunming mise immuned with the multivalent vaccine,while no specific fluorescence or brown particles appeared in muscle of the control groups.These results demonstrated the expression of the recombinant plasmids pVAX1/SjHGPRT, pVAX1/SjSDISP and pVAX1/SjHGPRTo SDISP were successful.
3.Compared with the NS group and pVAX1 group,both pVAX1/SjHGPRT·SDISP and monovalent vaccine pVAX1/SjSDISP、pVAX1/SjHGPRT group gained statistically significant protection result. Also statistically significant was induced compared to the monovalent vaccine group on reduction rate in uterus.In the same time statistically significant was attained between pVAX1/SjSDISP group and pVAX1/ SjHGPRT group.While the worm and the liver egg reduction rates between pVAX1/SjHGPRT·SDISP and monovalent vaccine group were no statistically significant.Among all the groups the egg reduction rates are higher than the worm reduction rates.
Conclusion
1.Both the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP are successfully constructed.Both the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP are successfully expressed in the quadriceps femoris of Kunming mise.
2.Both the monovalent vaccine pVAX1/SjHGPRT、pVAX1/SjSDISP and the multivalent vaccine pVAX1/SjHGPRT·SDISP can induced partial protection in mice.The multivalent vaccine pVAX1/SjHGPRT·SDISP can induce significant immunoprotection which is better than the monovalent vaccine on reduction rate in uterus.
引文
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