甲型流感病毒NP基因真核表達載體的構(gòu)建及其免疫效果研究
發(fā)布時間：2018-07-27 12:22
【摘要】： 目的 流行性感冒(簡稱流感)是由甲(A)、乙(B)、丙(C)三型流感病毒分別引起的急性呼吸道感染，其中甲型流感病毒在自然界中廣泛分布，常以流行形式出現(xiàn)，可引起世界性流感大流行，對人類健康和世界經(jīng)濟造成巨大影響。流感病毒在自然界中不僅能經(jīng)常發(fā)生抗原性漂移，而且也能發(fā)生同型毒株間基因片段的重配，這樣就造成了流感疫苗的時效性。因此，研制具有交叉保護能力的流感疫苗對于有效防止流感的發(fā)生和流行具有重要意義。 甲型流感病毒RNA分為8個節(jié)段，其中第5節(jié)段編碼其核衣殼蛋白(nucleoprotein，NP)。NP參與病毒復(fù)制周期的多個階段，影響病毒的轉(zhuǎn)錄、復(fù)制、裝配及轉(zhuǎn)運功能：此外，NP也是細胞毒性T淋巴細胞(cytotoxic T lymphocytes，CTL)識別的主要抗原，CTL通過識別MHC-Ⅰ類分子遞呈的核蛋白抗原肽而清除感染的流感病毒，因而能對不同亞型的病毒株產(chǎn)生交叉保護性。 本研究旨在構(gòu)建pcDNA3.1(+)／NP的核酸疫苗，觀察其在真核細胞中的表達，并在機體水平上研究其單獨應(yīng)用及與本室成功構(gòu)建的pcDNA3.1(+)／HA聯(lián)合應(yīng)用的抗流感病毒免疫效果。 方法 1．甲型流感病毒NP基因DNA疫苗的構(gòu)建：從流感病毒株A／PR／8／34(H1N1)感染的雞胚尿囊液中提取病毒RNA，用RT-PCR技術(shù)擴增NP基因，并將擴增的NP基因克隆入真核表達載體pcDNA3.1(+)，構(gòu)建NP基因的真核表達載體——pcDNA3.1(+)／NP。 2．pcDNA3.1(+)／NP的初步表達：將pcDNA3.1(+)／NP質(zhì)粒DNA用脂質(zhì)體介導(dǎo)法轉(zhuǎn)染HEK293細胞，然后用免疫熒光檢測重組質(zhì)粒的表達情況。再將pcDNA3.1(+)／NP質(zhì)粒DNA經(jīng)肌肉注射免疫動物，免疫組化技術(shù)檢測NP基因在肌細胞的表達及持續(xù)時間。 3．pcDNA3.1(+)／NP質(zhì)粒DNA的免疫效果研究：將pcDNA3.1(+)／NP質(zhì)粒DNA采用DNA疫苗的肌肉注射方式免疫動物，通過免疫檢測技術(shù)觀察其對機體的抗流感病毒免疫應(yīng)答；并用同一亞型流感病毒攻擊免疫小鼠，觀察肺指數(shù)和肺指數(shù)抑制率等指標；最后用不同亞型的流感病毒攻擊免疫小鼠，觀察保護率。綜合評價pcDNA3.1(+)／NP作為DNA疫苗的保護效果，和與pcDNA3.1(+)／HA聯(lián)合應(yīng)用的免疫效果。 結(jié)果 1．經(jīng)RT-PCR技術(shù)從流感病毒RNA中擴增到NP基因，經(jīng)酶切后插入pcDNA3.1(+)，構(gòu)建成pcDNA3.1(+)／NP。該重組載體經(jīng)限制性內(nèi)切酶酶切、PCR及序列測定等鑒定，證實pcDNA3.1(+)／NP構(gòu)建成功。 2．pcDNA3.1(+)／NP質(zhì)粒DNA轉(zhuǎn)染HEK293細胞后，免疫熒光結(jié)果顯示所轉(zhuǎn)染的細胞有較強的綠色熒光信號。給實驗動物肌肉內(nèi)注射pcDNA3.1(+)／NP質(zhì)粒DNA后，在實驗觀察的所有時間點均可觀測到NP蛋白的表達。 3．淋巴細胞增殖試驗表明，動物接種pcDNA3.1(+)／NP質(zhì)粒DNA后，在流感病毒抗原的刺激下，，能夠誘導(dǎo)有效的淋巴細胞增殖反應(yīng)。流感病毒攻擊后肺指數(shù)等指標顯示，pcDNA3.1(+)／NP質(zhì)粒DNA與pcDNA3.1(+)／HA質(zhì)粒DNA聯(lián)合使用對抑制肺病變程度的效果顯著。被pcDNA3.1(+)／NP質(zhì)粒DNA免疫的動物對同亞型流感病毒攻擊的保護率為50％，對不同亞型流感病毒攻擊的保護率為37.5％，而且與pcDNA3.1(+)／HA質(zhì)粒DNA聯(lián)合使用后可明顯提高保護率。 結(jié)論 1．成功構(gòu)建了甲型流感病毒NP基因的真核表達載體——pcDNA3.1(+)／NP。 2．免疫熒光技術(shù)證實了NP基因能夠在哺乳動物細胞中表達；實驗動物肌肉注射該重組質(zhì)粒DNA后可在肌細胞內(nèi)檢測到目的基因表達。 3．淋巴細胞增殖試驗證實，pcDNA3.1(+)／NP的質(zhì)粒DNA免疫動物后能有效誘導(dǎo)機體產(chǎn)生抗流感病毒的細胞免疫。pcDNA3.1(+)／NP的質(zhì)粒DNA免疫動物后，對同一亞型和／或不同亞型流感病毒的攻擊都能提供有效的保護作用，與pcDNA3.1(+)／HA聯(lián)合應(yīng)用可明顯提高保護效果。 綜上所述，提示pcDNA3.1(+)／NP可作為具有交叉保護作用的、抗流感病毒的侯選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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346;R392
本文編號：2147811
[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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346;R392
【參考文獻】
相關(guān)期刊論文 前4條
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2 陳則;五種編碼流感病毒蛋白的DNA疫苗的免疫原性研究[J];中國生物制品學(xué)雜志;2002年01期
3 喬傳玲,姜永萍,田國斌,鄧國華,王秀榮,陳化蘭,于康震;表達禽流感HA和NP雙基因重組禽痘病毒的免疫保護性[J];畜牧獸醫(yī)學(xué)報;2004年02期
4 喬傳玲,于康震,姜永萍,張建林,鄧國華,孟慶文,田國斌,唐秀英;表達禽流感病毒核蛋白基因重組禽痘病毒的構(gòu)建及其免疫保護性研究[J];中國農(nóng)業(yè)科學(xué);2003年06期
本文編號：2147811
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