本文關鍵詞： 冠狀病毒 SARS DNA免疫 細胞因子　出處：《新疆農(nóng)業(yè)大學》2006年碩士論文　論文類型：學位論文

【摘要】： SARS ( Severe Acute Respiratory Syndrome)是一種傳染性強、病死率高、嚴重危害人民生命健康的傳染病。世界衛(wèi)生組織于2003年4月6日宣布引起SARS的病原體是一種新的病毒,屬于冠狀病毒家族,稱為SARS相關冠狀病毒(SARS-Associated coronavirus, SARS-CoV )。SARS-CoV表現(xiàn)出許多不同于冠狀病毒的特性,為冠狀病毒中一個新的分支。目前,病毒性疾病尚無特效治療方法,對于SARS也是一樣。所以,控制SARS的最好辦法是預防,而疫苗的開發(fā)則是建立有效防范措施的關鍵環(huán)節(jié)。DNA疫苗是繼滅活疫苗、亞單位疫苗之后的第三代疫苗,具有許多的優(yōu)越性。所以本研究選擇了SARS病毒DNA疫苗這一方向,以SARS-CoV主要結構蛋白S、N、E和M蛋白基因作為研究對象。構建針對SARS-CoV結構蛋白基因的重組核酸疫苗,觀察其免疫小鼠后機體的特異性免疫應答的情況,探討其作為SARS-CoV疫苗的可能性。 本研究由兩部分組成: 第一部分為SARS-CoV結構蛋白基因重組核酸疫苗的構建。從GenBank上獲得SARS(BJ01)S、N、E和M蛋白基因的序列,使用引物設計軟件Primer6.0進行引物設計。以SARS-CoV(BJ01)cDNA為模板,合適條件下進行PCR,獲得SARS-CoV SARS-CoV S、N、E和M蛋白基因片段。經(jīng)酶切處理后,定向克隆于pVAX1真核表達載體,構建包含SARS-CoV S、N、E和M基因片段的重組質粒pVAX/S、pVAX/N、pVAX/E和pVAX/M。經(jīng)酶切和測序等方法鑒定重組質粒的正確性。然后利用間接免疫熒光法鑒定重組質粒的體外表達。將重組質粒瞬時轉染真核細胞。48小時后IFA試驗檢測目的基因的表達產(chǎn)物的活性。 第二部分為真核表達質粒pVAX/S、pVAX/N、pVAX/E和pVAX/M在小鼠體內(nèi)的免疫原性研究。將重組質粒按100ug/只肌肉注射BALB/c小鼠,第一次免疫后2周進行加強免疫,4周后進行第三次免疫。試驗分為空載體組,實驗組。在免疫后的2、4、6、8、10周分別采集并分離小鼠血清。用流式細胞儀檢測相關細胞因子TNF、IFN-?、IL-5、IL-4、Il-2水平。 實驗結果顯示,重組質粒DNA免疫小鼠后能夠誘導機體產(chǎn)生特異的細胞免疫和體液免疫應答。這為進一步研制安全、有效的SARS DNA疫苗奠定了基礎。
[Abstract]:SARS (Severe Acute Respiratory Syndromeis) is a kind of infectious disease with high mortality and serious harm to people's life and health. In April 6th 2003, the World Health Organization declared that the pathogen causing SARS is a new virus, belonging to the coronavirus family. Called SARS associated coronavirus. SARS-CoV coronavirus.SARS-CoV shows many characteristics different from coronavirus.It is a new branch of coronavirus. At present, there is no special treatment for viral diseases, and the same is true for SARS. The best way to control SARS is to prevent it, and the development of vaccine is the key link to establish effective preventive measures. DNA vaccine is the third generation vaccine after inactivated vaccine and subunit vaccine. Therefore, we chose the direction of DNA vaccine of SARS virus to construct recombinant nucleic acid vaccine against SARS-CoV structural protein gene. To observe the specific immune response of mice immunized with it and to explore the possibility of its use as SARS-CoV vaccine. This study consists of two parts:. The first part is the construction of recombinant nucleic acid vaccine for structural protein gene of SARS-CoV. The sequence of SARS-CoV BJ01SJ01SN and M protein gene was obtained from GenBank. Primer design software Primer6.0 was used for primer design. SARS-CoV(BJ01)cDNA was used as template. Under suitable conditions, SARS-CoV SARS-CoV signon E and M protein fragments were obtained and cloned into pVAX1 eukaryotic expression vector after restriction endonuclease digestion. The recombinant plasmids pVAX / SapVAX / pVAXP / NV AXR / E and pVAXR / M were constructed. The correctness of the recombinant plasmids was confirmed by restriction endonuclease digestion and sequencing. The in vitro expression of the recombinant plasmids was identified by indirect immunofluorescence assay. The transient expression of the recombinant plasmids was determined. The activity of the expressed products of the target gene was detected by IFA assay after transfection of eukaryotic cells. In the second part, the immunogenicity of the eukaryotic expression plasmid pVAX- pVAX / NV AX / NV AXP / E and pVAX/M in mice was studied. The recombinant plasmid was injected intramuscularly into BALB/c mice. The experiment was divided into empty carrier group and experimental group. Serum samples were collected and isolated from mice at 10 weeks after immunization. TNFF IFN-IFN was detected by flow cytometry. IL-5, IL-4 and Il-2 levels. The results showed that the recombinant plasmid DNA could induce specific cellular and humoral immune responses in mice, which laid a foundation for the further development of a safe and effective SARS DNA vaccine.
【學位授予單位】：新疆農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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