| 罗四维1 汪世平2.抗日本血吸虫pVAX1/ SjHGPRT·SDISP 多价疫苗的构建方法探讨[J].,2011,11(19):3629-3631 |
| 抗日本血吸虫pVAX1/ SjHGPRT·SDISP 多价疫苗的构建方法探讨 |
| Construction Method of Multivalent DNA Vaccine pVAX1/ SjHGPRT·SDISP Against Schistosoma Japonicum |
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| DOI： |
| 中文关键词: 血吸虫 pVAX1/ SjHGPRT·SDISP 多价疫苗 构建 |
| 英文关键词: Schistosoma japonicum PVAX1/ SjHGPRT·SDISP Multivalent vaccine Construction |
| 基金项目:国家重点基础研究发展（973）计划（No. 2007CB513108）；国家科技支撑计划（No. 2009BAI78B05）；
湖南省血吸虫病免疫与传播控制重点实验室、湖南省重点学科建设专项经费（No. 07-985-2） |
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| 中文摘要: |
| 目的：探讨抗日本血吸虫生殖产卵编码基因多价疫苗pVAX1/ SjHGPRT·SDISP 的构建方法并从基因与蛋白水平验证该构
建方法是否成功。方法：分别以pcDNA3.0/SjHGPRT、pcDNA3.0/SjSDISP 为模板，设计引物扩增SjHGPRT、SjSDISP，采用overlap
方法扩增全长SjHGPRT·SDISP。将纯化后的产物SjHGPRT·SDISP 以及pVax1 质粒采用Kpn I 和Xba I 双酶切，1%低熔点胶回
收目的DNA 片段以及酶切后Pvax1 载体片段双胶连。连接产物转化至DH5α 细胞。挑选阳性克隆采用双酶切以及测序方法从
基因水平鉴定是否构建成功。将构建产物免疫观察动物，采用免疫组化方法从蛋白水平鉴定疫苗pVAX1/ SjHGPRT·SDISP 是否
构建成功。结果：酶切方法以及测序结果均显示重组质粒的插入序列分别与目的基因完全一致，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。结论：真核重组质粒pVAX1/SjHG- PRT·
SDISP 构建成功，且在昆明鼠肌肉细胞内能够获得良好的表达。 |
| 英文摘要: |
| Objective: To explore the method to construct the multivalent DNA vaccine pVAX1/ SjHGPRT·SDIS and certify
whether this method was successful at both gene and protein level. Methods: SjHGPRT and SjSDISP were amplified with the templates
of pcDNA3.0/SjHGPRT and pcDNA3.0/SjSDISP. Full-length SjHGPRT·SDISP were amplified by overlap method. The purified products
SjHGPRT·SDISP and plasmid pVax1 were digested with Kpn I andXba I. 1% low melting Gel was used to collect the fragments of
DNA and make the carriers' fragments of Pvax1 glue together after digestion. The ligated product was transformed into DH5α cell. Positive
clones were screened to confirm whether the construction is successful at gene level with the method of digestion and sequencing.
Use the products of construction to immune and observe animals and confirm whether the construction of vaccine pVAX1/ SjHGPRT·SDISP
was successful with the method of immunohistochemical staining. Results: Restriction enzyme digestion and sequencing results
showed that the inserted sequence of the recombinant plasmid is completely the same with the target gene. 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. Conclusions: The pVAX1/ SjHGPRT·SDISP was successfully
constructed, which may be well expressed in the muscle of vaccined mice. |
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