mBD2与HA1融合基因真核表达载体的构建及其免疫保护性研究

发布时间：2018-05-06 16:06

本文选题：流感病毒 + 血凝素　；参考：《四川大学》2007年硕士论文

【摘要】： 目的甲型流感病毒曾在世界上多次发生大规模的流感暴发流行，目前对流感尚无特效药物进行治疗，接种流感疫苗是易感者预防流感的最佳选择，核酸疫苗是当前流感疫苗研究的方向与热点。流感病毒血凝素(hemagglutinin，HA)是流感病毒的主要保护性抗原，能刺激机体产生中和性抗体，此类抗体具有中和病毒和防止病毒进入靶细胞的作用，可中断病毒的复制。HA由HA1重链和HA2轻链相连构成。HA的5个抗原决定簇主要都位于HA1区，因而流感亚单位疫苗的靶标就首选HA1区。小鼠β-防御素2(mBD2)是一种内源性的抗菌肽，对非成熟的DC细胞具有趋化作用，能活化APC，，增强机体的免疫作用，因此可以作为一种天然的免疫佐剂。本研究的目的在于构建mBD2与HA1融合的真核表达载体(pcDNA3.1(+)／mBD2-HA1)，并检测其在HEK293细胞中的表达，观察pcDNA3.1(+)／mBD2-HA1免疫小鼠后对流感病毒攻击的保护力，研究mBD2作为免疫佐剂的可行性。方法用PCR技术分别从构建好的质粒pcDNA3.1(+)／mBD2和pcDNA3.1(+)／HA1中扩增出mBD2与HA1基因片段，再用重叠延伸PCR法(overlap-PCR)将mBD2与HA1通过一段柔性多肽接头Gly_4Ser融合为mBD2—HA1。将mBD2—HA1融合基因插入真核表达载体pcDNA3.1(+)，转化感受态E. coli JM109并筛选阳性克隆。经酶切、PCR和测序鉴定正确后，用脂质体将重组质粒pcDNA3.1(+)／mBD2-HA1转染HEK293细胞，用课题组制备的兔抗流感病毒血清作为一抗，FITC荧光素标记的羊抗兔IgG为二抗，通过免疫荧光检测融合蛋白的真核表达情况。为了研究重组质粒pcDNA3.1(+)／mBD2-HA1对甲型流感病毒的免疫保护作用，我们以SPF级BALB／c小鼠为实验动物，用pcDNA3.1(+)／mBD2-HA1、pcDNA3.1(+)／HA1和pcDNA3.1(+)质粒DNA分别免疫小鼠，一共免疫两次，每次间隔三周。在第二次加强免疫前，用断尾法从小鼠尾部取血；第二次免疫后10d(病毒攻击后3d)，再次取血，并收集血清检测抗-HA抗体。加强免疫后一周，用致死量(10×LD50)滴鼻攻击小鼠，观测动物的体重变化及存活率。综合以上几个方面评价重组质粒的免疫保护效果。结果 PCR法分别从质粒pcDNA3.1(+)／mBD2和pcDNA3.1(+)／HA1中扩增出了mBD2与HA1基因片段，并且用重叠延伸PCR法成功将mBD2与HA1通过一段柔性多肽接头Gly_4Ser融合为mBD2-HA1。重组质粒pcDNA3.1(+)／mBD2-HA1经双酶切电泳后出现大小约1.2kb的目的条带，PCR鉴定也出现了相同大小的条带，目的片段经测序和将测序结果进行Blast比对，证实了克隆片段是正确的。免疫荧光检测结果表明重组质粒pcDNA3.1(+)／mBD2-HA1体外实验能够在HEK293细胞中表达mBD2-HA1的融合蛋白。动物实验结果表明，pcDNA3.1(+)空质粒对照组的小鼠7d内全部死亡，pcDNA3.1(+)／HA1免疫组的小鼠保护率为50％，而pcDNA3.1(+)／mBD2-HA1组的小鼠保护率为90％；与单独免疫pcDNA3.1(+)／HA1相比，mBD2-HA1融合基因DNA免疫小鼠后，诱导出了更高的抗-HA抗体，保护率也更高，并且体重丢失明显减少。结论综上所述，本研究成功构建了mBD2—HA1融合基因的真核表达质粒pcDNA3.1(+)／mBD2-HA1，该质粒能在真核细胞中表达融合蛋白；经动物实验表明，pcDNA3.1(+)／mBD2-HA1重组质粒DNA免疫实验动物后，能够为抗流感病毒攻击提供很好的保护力，其保护效果明显高于pcDNA3.1(+)／HA1免疫组。本研究结果初步表明：小鼠β防御素2可以作为一种免疫佐剂用于流感病毒的DNA疫苗中。
[Abstract]:Purpose

Influenza A virus ( HA ) is the main protective antigen of influenza virus , which is the main protective antigen of influenza virus , which can stimulate organism to produce neutralizing antibody . The HA is composed of HA1 heavy chain and HA2 light chain .

The aim of this study was to construct the eukaryotic expression vector ( pcDNA3.1 ( + ) / mBD2 - HA1 ) fused with mBD2 and HA1 , and to detect the expression of pcDNA3.1 ( + ) / mBD2 - HA1 , and to observe the protective effect of pcDNA3.1 ( + ) / mBD2 - HA1 on influenza virus attack , and to study the feasibility of mBD2 as an adjuvant .

method

The recombinant plasmid pcDNA3.1 ( + ) / mBD2 - HA1 was inserted into eukaryotic expression vector pcDNA3.1 ( + ) . The recombinant plasmid pcDNA3.1 ( + ) / mBD2 - HA1 was inserted into eukaryotic expression vector pcDNA3.1 ( + ) .
After 10 days after the second immunization ( 3 days after the challenge of the virus ) , the blood was taken again and the anti - HA antibody was collected . One week after immunization , the mice were challenged with lethal dose ( 10 脳 LD50 ) to observe the weight change and survival rate of the animals . The immune protective effect of the recombinant plasmid was evaluated in the above aspects .

Results

mBD2 and HA1 were amplified from pcDNA3.1 ( + ) / mBD2 and pcDNA3.1 ( + ) / HA1 by PCR , and mBD2 and HA1 were fused to mBD2 - HA1 by overlapping extension PCR . The recombinant plasmid pcDNA3.1 ( + ) / mBD2 - HA1 was subjected to blast - specific electrophoresis . The results showed that the recombinant plasmid pcDNA3.1 ( + ) / mBD2 - HA1 was able to express mBD2 - HA1 in vitro . The results showed that the protective rate of pcDNA3.1 ( + ) / mBD2 - HA1 was 50 % , while the protective rate of pcDNA3.1 ( + ) / mBD2 - HA1 was 90 % .
Compared with pcDNA3.1 ( + ) / HA1 alone , mBD2 - HA1 fusion gene DNA immunized mice induced higher anti - HA antibody , higher protection rate , and decreased body weight loss .

Conclusion

In conclusion , the eukaryotic expression plasmid pcDNA3.1 ( + ) / mBD2 - HA1 of mBD2 - HA1 fusion gene was successfully constructed .
The protective effect of pcDNA3.1 ( + ) / mBD2 - HA1 was significantly higher than that of pcDNA3.1 ( + ) / HA1 .

【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】