甲型流感病毒NP基因真核表达载体的构建及其免疫效果研究

发布时间：2018-07-27 12:22

【摘要】： 目的流行性感冒(简称流感)是由甲(A)、乙(B)、丙(C)三型流感病毒分别引起的急性呼吸道感染，其中甲型流感病毒在自然界中广泛分布，常以流行形式出现，可引起世界性流感大流行，对人类健康和世界经济造成巨大影响。流感病毒在自然界中不仅能经常发生抗原性漂移，而且也能发生同型毒株间基因片段的重配，这样就造成了流感疫苗的时效性。因此，研制具有交叉保护能力的流感疫苗对于有效防止流感的发生和流行具有重要意义。甲型流感病毒RNA分为8个节段，其中第5节段编码其核衣壳蛋白(nucleoprotein，NP)。NP参与病毒复制周期的多个阶段，影响病毒的转录、复制、装配及转运功能：此外，NP也是细胞毒性T淋巴细胞(cytotoxic T lymphocytes，CTL)识别的主要抗原，CTL通过识别MHC-Ⅰ类分子递呈的核蛋白抗原肽而清除感染的流感病毒，因而能对不同亚型的病毒株产生交叉保护性。本研究旨在构建pcDNA3.1(+)／NP的核酸疫苗，观察其在真核细胞中的表达，并在机体水平上研究其单独应用及与本室成功构建的pcDNA3.1(+)／HA联合应用的抗流感病毒免疫效果。方法 1．甲型流感病毒NP基因DNA疫苗的构建：从流感病毒株A／PR／8／34(H1N1)感染的鸡胚尿囊液中提取病毒RNA，用RT-PCR技术扩增NP基因，并将扩增的NP基因克隆入真核表达载体pcDNA3.1(+)，构建NP基因的真核表达载体——pcDNA3.1(+)／NP。 2．pcDNA3.1(+)／NP的初步表达：将pcDNA3.1(+)／NP质粒DNA用脂质体介导法转染HEK293细胞，然后用免疫荧光检测重组质粒的表达情况。再将pcDNA3.1(+)／NP质粒DNA经肌肉注射免疫动物，免疫组化技术检测NP基因在肌细胞的表达及持续时间。 3．pcDNA3.1(+)／NP质粒DNA的免疫效果研究：将pcDNA3.1(+)／NP质粒DNA采用DNA疫苗的肌肉注射方式免疫动物，通过免疫检测技术观察其对机体的抗流感病毒免疫应答；并用同一亚型流感病毒攻击免疫小鼠，观察肺指数和肺指数抑制率等指标；最后用不同亚型的流感病毒攻击免疫小鼠，观察保护率。综合评价pcDNA3.1(+)／NP作为DNA疫苗的保护效果，和与pcDNA3.1(+)／HA联合应用的免疫效果。结果 1．经RT-PCR技术从流感病毒RNA中扩增到NP基因，经酶切后插入pcDNA3.1(+)，构建成pcDNA3.1(+)／NP。该重组载体经限制性内切酶酶切、PCR及序列测定等鉴定，证实pcDNA3.1(+)／NP构建成功。 2．pcDNA3.1(+)／NP质粒DNA转染HEK293细胞后，免疫荧光结果显示所转染的细胞有较强的绿色荧光信号。给实验动物肌肉内注射pcDNA3.1(+)／NP质粒DNA后，在实验观察的所有时间点均可观测到NP蛋白的表达。 3．淋巴细胞增殖试验表明，动物接种pcDNA3.1(+)／NP质粒DNA后，在流感病毒抗原的刺激下，，能够诱导有效的淋巴细胞增殖反应。流感病毒攻击后肺指数等指标显示，pcDNA3.1(+)／NP质粒DNA与pcDNA3.1(+)／HA质粒DNA联合使用对抑制肺病变程度的效果显著。被pcDNA3.1(+)／NP质粒DNA免疫的动物对同亚型流感病毒攻击的保护率为50％，对不同亚型流感病毒攻击的保护率为37.5％，而且与pcDNA3.1(+)／HA质粒DNA联合使用后可明显提高保护率。结论 1．成功构建了甲型流感病毒NP基因的真核表达载体——pcDNA3.1(+)／NP。 2．免疫荧光技术证实了NP基因能够在哺乳动物细胞中表达；实验动物肌肉注射该重组质粒DNA后可在肌细胞内检测到目的基因表达。 3．淋巴细胞增殖试验证实，pcDNA3.1(+)／NP的质粒DNA免疫动物后能有效诱导机体产生抗流感病毒的细胞免疫。pcDNA3.1(+)／NP的质粒DNA免疫动物后，对同一亚型和／或不同亚型流感病毒的攻击都能提供有效的保护作用，与pcDNA3.1(+)／HA联合应用可明显提高保护效果。综上所述，提示pcDNA3.1(+)／NP可作为具有交叉保护作用的、抗流感病毒的侯选DNA疫苗。
[Abstract]:objective
Influenza (influenza) is an acute respiratory infection caused by influenza A (A), (B), C (C) influenza virus respectively. Influenza A virus is widely distributed in nature, often in the form of epidemic, causing a worldwide influenza pandemic and has a great impact on human health and the world economy. Influenza virus is in nature. Not only can the excursion of antigenicity often occur, but also the redistribution of gene fragments between the same type of virus can also occur, which leads to the timeliness of influenza vaccine. Therefore, the development of a cross protective influenza vaccine is of great significance to prevent the occurrence and epidemic of influenza.
Influenza A virus RNA is divided into 8 segments, of which fifth segments encoded its nucleoprotein (NP).NP to participate in multiple stages of the virus replication cycle, affecting the transcription, replication, assembly and transport functions of the virus: in addition, NP is also the main antigen identified by the cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTL), CTL by identifying M HC- class I molecules express the nucleoprotein antigen peptide and eliminate influenza virus infection, so that they can produce cross protection for different subtypes of virus strains.
The aim of this study was to construct a pcDNA3.1 (+) / NP nucleic acid vaccine, to observe its expression in eukaryotic cells, and to study the anti influenza virus immune effect of the combined application of pcDNA3.1 (+) / HA combined with the successful construction of this chamber on the level of the body.
Method
1. DNA vaccine of influenza A virus NP gene: extracting virus RNA from the chicken embryo allantoic fluid infected by influenza virus strain A / PR / 34 (H1N1), amplifying NP gene by RT-PCR technique and cloning the amplified NP gene into eukaryotic expression vector pcDNA3.1 (+), and constructing the eukaryotic expression vector of NP gene -- pcDNA3.1 (+) / Parliament
The initial expression of 2.pcDNA3.1 (+) / NP: transfection of pcDNA3.1 (+) / NP plasmid DNA with liposome mediated HEK293 cells, and then using immunofluorescence to detect the expression of recombinant plasmids. Then pcDNA3.1 (+) / NP plasmid DNA was injected into the animals by intramuscular injection, and the expression and duration of NP gene in myocytes were detected by immunohistochemical technique.
Study on the immune effect of 3.pcDNA3.1 (+) / NP plasmid DNA: immunize animals with pcDNA3.1 (+) / NP plasmid DNA using DNA vaccine and observe the immune response to influenza virus by immunoassay, and attack the mice with the same subtype of influenza virus, and observe the index of lung index and lung index inhibition rate. At last, the immunized mice were attacked with different subtypes of influenza virus to observe the protection rate. The protective effect of pcDNA3.1 (+) / NP as the DNA vaccine and the immune effect combined with pcDNA3.1 (+) / HA were evaluated synthetically.
Result
1. the NP gene was amplified from influenza virus RNA by RT-PCR, and pcDNA3.1 (+) was inserted after the enzyme digestion. The recombinant vector of pcDNA3.1 (+) / NP. was constructed by restriction endonuclease digestion, PCR and sequence determination, which confirmed that the construction of pcDNA3.1 (+) / NP was successful.
After transfection of 2.pcDNA3.1 (+) / NP plasmid DNA to HEK293 cells, the immunofluorescence results showed that the transfected cells had strong green fluorescence signals. After intramuscular injection of pcDNA3.1 (+) / NP plasmid DNA to the experimental animals, the expression of NP protein could be observed at all time points of the experimental observation.
The 3. lymphocyte proliferation test showed that after the inoculation of pcDNA3.1 (+) / NP plasmid DNA, the effective lymphocyte proliferation reaction could be induced under the stimulation of influenza virus antigen. The index of lung index after influenza virus attack showed that the combination of pcDNA3.1 (+) / NP plasmid DNA and pcDNA3.1 (+) / HA plasmid was used to inhibit the degree of lung disease. The protection rate of the animals immunized with pcDNA3.1 (+) / NP plasmid DNA was 50%, the protection rate for different subtype influenza virus attacks was 37.5%, and the protection rate could be obviously improved after the combination of pcDNA3.1 (+) / HA plasmid DNA.
conclusion
1. successfully constructed the eukaryotic expression vector of influenza A virus NP gene pcDNA3.1 (+) / NP.
2. immunofluorescence technique confirmed that the NP gene could be expressed in the mammalian cells, and the recombinant plasmid DNA could be detected in the muscle cells after the intramuscular injection of the recombinant plasmid.
The 3. lymphocyte proliferation test confirmed that pcDNA3.1 (+) / NP plasmid DNA immunized animals effectively to induce immunization of.PcDNA3.1 (+) / NP plasmid DNA immune animals, which could provide effective protection against the attack of the same subtype and / or different subtype of influenza virus, and combined with pcDNA3.1 (+) / HA. It can obviously improve the protection effect.
In conclusion, pcDNA3.1 (+) / NP can be used as a cross protection DNA vaccine against influenza virus.
【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】：R346;R392

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