本文選題：脊髓灰質(zhì)炎疫苗Sabin + 1株病毒�。� 參考：《第四軍醫(yī)大學(xué)》2007年博士論文

【摘要】： 流感病毒RNA聚合酶缺少校正功能,導(dǎo)致基因組在復(fù)制過程中容易發(fā)生突變,人群中因以往感染產(chǎn)生的抗體不能有效地防止新毒株感染,因而每年需要注射流感疫苗,給社會(huì)帶來沉重的經(jīng)濟(jì)負(fù)擔(dān)。如果有一種通用型疫苗,經(jīng)過全程免疫后,產(chǎn)生廣譜性抗體,則能夠預(yù)防各種變異病毒,或降低感染病毒后疾病的嚴(yán)重程度。甲型流感病毒囊膜上除血凝素(HA)和神經(jīng)氨酸酶(NA)外,還含有一個(gè)分子量較小的基質(zhì)蛋白M2,其胞外區(qū)(M2e)非常保守,是研制通用型疫苗的最佳靶位。由于M2e只含有24個(gè)氨基酸,需要以各種措施來增強(qiáng)其免疫原性。本論文擬將口服脊髓灰質(zhì)炎疫苗(OPV)Sabin1株病毒改造為疫苗載體,采用多聚蛋白融合策略,插入流感M2e基因,將前者作為表達(dá)載體和免疫佐劑,提高人體針對(duì)M2e的免疫應(yīng)答,構(gòu)建一種能抵御各種甲型流感病毒的通用型疫苗。 通過RT-PCR方法擴(kuò)增了Sabin1株病毒基因組的三個(gè)片段f1、f2、f3并分別克隆,在f1和f2、f2和f3片段之間含重疊序列,內(nèi)有基因組唯一的酶切位點(diǎn)。因此,三片段可以通過酶切和連接獲得全基因組。將三個(gè)克隆分別測序,與GenBank中序列比較,發(fā)現(xiàn)在基因組7440bp中只有三個(gè)位點(diǎn)不同,其中26 A→G,355 C→T兩個(gè)位點(diǎn)處于非編碼區(qū),6735 A→G導(dǎo)致聚合酶3D上250位氨基酸由賴氨酸變?yōu)榫彼�。說明Sabin1株基因組比較穩(wěn)定,適合于作為疫苗載體。 基因組起始密碼子(nt743)位于pGEM-f1質(zhì)粒上,通過重疊PCR方法在之前插入多克隆位點(diǎn)(EcoRI、XmaI、XhoI)和人工蛋白酶切位點(diǎn)(ALFQG)構(gòu)成的功能盒(Cassette),形成pGEM-f1m質(zhì)粒;同樣,在位于pGEM-f2質(zhì)粒上P1/P2結(jié)合處引入類似序列,構(gòu)建為pGEM-f2m質(zhì)粒。連接為基因組后可形成兩種基本型載體PV1和PV2。多克隆位點(diǎn)內(nèi)插入的外源基因隨多聚蛋白表達(dá)后,在病毒編碼的蛋白酶切割后釋放外源蛋白。 為提高載體RNA的轉(zhuǎn)染效果,另外在載體上進(jìn)行兩項(xiàng)改造:基因組前加入錘頭型核酶(Rz)基序,合成RNA后發(fā)揮核酶功能,去除多余核苷酸,產(chǎn)生PV基因組真正的5’-末端;在基因組后導(dǎo)入poly(A)40結(jié)構(gòu),后者是PV RNA感染的必要基序。兩個(gè)基序均以PCR引物延伸方法產(chǎn)生,并通過基因置換方式導(dǎo)入到pGEM-f1m和pGEM-f3中,經(jīng)測序證明引入的基序與設(shè)計(jì)序列一致。將改造前和改造后的質(zhì)粒進(jìn)行三片段連接,形成八種載體,分別含有不同的功能盒及基序組合。 將攜帶各種PV載體的質(zhì)粒線性化后,以T7 RNA聚合酶體外轉(zhuǎn)錄法合成RNA并純化,通過電泳發(fā)現(xiàn)RNA完整性、均一性良好,適合于轉(zhuǎn)染細(xì)胞。對(duì)含有Rz序列的RNA進(jìn)行體外切割,經(jīng)PAGE電泳檢測反應(yīng)進(jìn)程,確定在合適的緩沖液中作用1.5小時(shí)獲得最佳切割效果。 采用電穿孔、脂質(zhì)體轉(zhuǎn)染法和磷酸鈣轉(zhuǎn)染法將提取的PV Sabin1 RNA轉(zhuǎn)染入Vero細(xì)胞,以形成的噬斑數(shù)目比較轉(zhuǎn)染效率,篩選轉(zhuǎn)染大分子RNA的最佳方法。實(shí)驗(yàn)結(jié)果證明脂質(zhì)體Tfx-20的轉(zhuǎn)染效率最佳,達(dá)到1.1~1.5×103感染性克隆/μg RNA,對(duì)細(xì)胞毒性不明顯。以Tfx-20轉(zhuǎn)染八種載體RNA,只有同時(shí)含有poly(A) 40和Rz基序的載體pPV1RzA和pPV2RzA能有效產(chǎn)生活病毒,約500~800克隆/μg RNA。感染性病毒產(chǎn)生時(shí)間與轉(zhuǎn)染RNA量有關(guān),當(dāng)以10μgRNA轉(zhuǎn)染時(shí),60小時(shí)就可以觀察到細(xì)胞病變;而以1μgRNA轉(zhuǎn)染時(shí),需要4天產(chǎn)生病變效應(yīng)。只含有poly(A)40基序的載體RNA也可以成功轉(zhuǎn)染,但效率低,當(dāng)RNA量為1μg時(shí)無法得到活病毒。不含兩個(gè)基序或只含Rz基序的RNA在Vero細(xì)胞上無感染性,即使將RNA量增加到30μg,培養(yǎng)10天亦無活病毒產(chǎn)生。 從霍亂弧菌中擴(kuò)增霍亂毒素B亞單位(CTB)基因,測序證明正確后,以PCR引物延伸法構(gòu)建CTB和M2e的融合基因,并在兩端分別添加EcoRI和XhoI位點(diǎn),通過雙酶切將融合基因?qū)氲絧PV1RzA中,保持正常的編碼框架,以建立的方法轉(zhuǎn)染Vero細(xì)胞,獲得了重組病毒rPV-CTBM2e。另外,擴(kuò)增SARS-冠狀病毒S蛋白的受體結(jié)合區(qū)基因S-RBD后,以同樣方法獲得了重組病毒rPV-S-RBD。提取重組病毒RNA,以RT-PCR方法擴(kuò)增涵蓋插入序列的片段并測序,證明兩個(gè)重組病毒均攜帶預(yù)期片段。 將重組病毒傳代多次,通過RT-PCR方法考察重組病毒的穩(wěn)定性。rPV-CTBM2e病毒傳代12次后仍保持完整的外源基因,而rPV-S-RBD病毒在傳代過程中丟失外源基因。rPV-CTBM2e病毒一步生長曲線證明其繁殖有延遲現(xiàn)象,但最終能得到和Sabin1病毒同樣的滴度。 將M2e合成多肽和KLH載體蛋白偶聯(lián)后免疫Balb/c小鼠,制備了針對(duì)M2e多肽的單克隆抗體,以ELISA方法篩選得到7株有活性的單抗。以單抗上清檢測感染Vero細(xì)胞的流感病毒,發(fā)現(xiàn)其中5株能與細(xì)胞表面的天然M2e結(jié)合,其中兩株為強(qiáng)陽性,可用于重組病毒M2e表達(dá)的檢測。 將重組病毒感染Vero細(xì)胞,通過免疫熒光方法,分別以抗M2e單抗和抗SARS-病毒免疫血清檢測,發(fā)現(xiàn)在細(xì)胞中出現(xiàn)明顯的熒光信號(hào),并集中在細(xì)胞質(zhì)中,說明外源蛋白在細(xì)胞質(zhì)中成功表達(dá),與預(yù)期結(jié)果一致。本研究為研制流感通用型疫苗和SARS-CoV疫苗奠定了基礎(chǔ)。
[Abstract]:Influenza virus RNA polymerase is lack of correction function, which causes the genome to mutate easily during the replication process. The antibody produced by the previous infection can not effectively prevent the infection of the new virus. Therefore, the influenza vaccine is injected every year to bring a heavy economic burden to the society. If there is a general type vaccine, after the whole course of immunization, The production of broad-spectrum antibodies can prevent a variety of variant viruses, or reduce the severity of the disease after infection. In addition to HA and NA, the envelope of influenza A virus also contains a small molecular weight matrix protein M2, and its extracellular domain (M2e) is very conservative. It is the best target for the development of universal vaccine. Because M2e With only 24 amino acids, it is necessary to enhance its immunogenicity with various measures. This paper intends to transform the oral poliomyelitis vaccine (OPV) Sabin1 strain virus into a vaccine carrier, and inserts the M2e gene into the influenza virus by using the fusion strategy of polyprotein, the former as an expression vector and an immune adjuvant, to improve the immune response to M2e, and to construct a kind of immune response to the human body. A universal vaccine that can resist all kinds of influenza A virus.
Three fragments of Sabin1 virus genome were amplified by RT-PCR method, F1, F2, F3 and cloned respectively. There were overlapping sequences between F1 and F2, F2 and F3 fragments, and the only enzyme cut site was in the genome. Therefore, the whole genome was obtained by the three fragment by enzyme cut and connection. The three clones were sequenced and found in the GenBank sequence. There are only three different loci in genomic 7440bp, of which 26 A, G and 355 C to T are in the non coding region. 6735 A to G causes the 250 amino acids on the polymerase 3D to arginine. It shows that the genome of the Sabin1 strain is more stable and is suitable for the vaccine carrier.
The genomic initiation codon (nt743) is located on the pGEM-f1 plasmid. The function box (Cassette), which is composed of EcoRI, XmaI, XhoI, and ALFQG, is inserted before the PCR method to form a pGEM-f1m plasmid. Similarly, a similar sequence is introduced into the P1/P2 binding site on the pGEM-f2 plasmid, which is constructed as a pGEM-f2m plasmid. After being inserted into the genome, the exogenous genes inserted in the polyclonal loci of the two basic carriers, PV1 and PV2., were expressed with the polyproteins, and the exogenous proteins were released after the virus encoded protease was cut.
In order to improve the transfection effect of carrier RNA, two modifications were carried out on the carrier: before the genome was added to the hammer head ribozyme (Rz), the function of the ribozyme was synthesized and the redundant nucleotides were removed and the true 5 '- terminal of the PV genome was produced; the poly (A) 40 structure was introduced after the genome, and the latter was the necessary order of PV RNA infection. Two sequences were all P The CR primer extension method was produced and introduced into pGEM-f1m and pGEM-f3 by gene replacement. The sequence was sequenced to agree with the design sequence. The modified and reformed plasmids were connected by three fragments to form eight carriers and contain different functional boxes and sequence combinations respectively.
After linearizing the plasmids carrying various PV carriers, RNA was synthesized by T7 RNA polymerase in vitro transcription. The integrity of RNA was found by electrophoresis, and the homogeneity was good. It was suitable for transfection of cells. The RNA in Rz sequence was cut in vitro and the reaction process was detected by PAGE electrophoresis. It was determined that the best effect was obtained in the appropriate buffer solution for 1.5 hours. Cutting effect.
The extracted PV Sabin1 RNA was transfected into Vero cells by electroporation, liposome transfection and calcium phosphate transfection. The best method of transfection of large molecule RNA was obtained by comparing the number of plaques formed by the number of plaque. The results showed that the transfection efficiency of liposome Tfx-20 was the best, reaching 1.1 ~1.5 x 103 infectious clones / micronux RNA. It is obvious that the transfection of eight carrier RNA with Tfx-20, only the carrier pPV1RzA and pPV2RzA containing poly (A) 40 and Rz motif, can produce a living virus effectively, and the time for the production of 500~800 cloned / Muu RNA. infective virus is related to the RNA quantity of transfection. When the transfection is 10 Mu gRNA, the cell lesion can be observed in 60 hours; and it takes 4 days when the transfection is 1 mu. The vector RNA that contains only poly (A) 40 can also be transfected successfully, but the efficiency is low. When the amount of RNA is 1 mu g, the live virus can not be obtained. No two motif or only Rz based RNA is not infected on Vero cells. Even if RNA is increased to 30 mu G, no live virus is produced for 10 days.
The gene of cholera toxin B subunit (CTB) was amplified from Vibrio cholerae. After the sequencing proved to be correct, the fusion gene of CTB and M2e was constructed by PCR primer extension method, and EcoRI and XhoI loci were added at both ends respectively. The fusion gene was introduced into pPV1RzA by double enzyme cutting, and the normal coding frame was maintained. The transfection of Vero cells was obtained by the method of establishment. Recombinant virus rPV-CTBM2e., after amplification of the receptor binding region gene S-RBD of the SARS- coronavirus S protein, obtained the recombinant virus rPV-S-RBD. from the recombinant virus rPV-S-RBD. to extract the recombinant virus RNA, and amplified the inserted sequence by RT-PCR method and sequenced it. It was proved that all the two recombinant viruses carried the expected fragment.
The recombinant virus was passed on several times, and the RT-PCR method was used to investigate the stability of the recombinant virus.RPV-CTBM2e virus for 12 times, and the whole foreign gene remained intact after 12 times, while the one step growth curve of the exogenous gene.RPV-CTBM2e virus was lost during the passage of the virus, which proved that the propagation of the recombinant virus was delayed, but eventually it could be similar to that of the Sabin1 virus. The titer.
After coupling M2e synthetic polypeptide and KLH carrier protein to immunize Balb/c mice, a monoclonal antibody against M2e polypeptide was prepared, and 7 active McAbs were screened by ELISA method. The detection of influenza virus in Vero cells was detected by the monoclonal antibody supernatant. It was found that 5 of them were capable of combining with the cell surface of Tian ran M2e, of which two were strongly positive and could be used for heavy weight. Detection of M2e expression in group virus.
The recombinant virus infected Vero cells was detected by immunofluorescence method and detected by anti M2e monoclonal antibody and anti SARS- virus sera respectively. It was found that the obvious fluorescent signal was found in the cell and concentrated in the cytoplasm, indicating that the exogenous protein was successfully expressed in the cytoplasm, which was the same as expected fruit. This study is to develop a general influenza vaccine and SARS. The -CoV vaccine lays the foundation.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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