本文關(guān)鍵詞： 豬3型腺病毒 慢病毒 穩(wěn)定細(xì)胞系 病毒增殖與包裝　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬3型腺病毒(PAdV-3)于1964年從健康豬腸道首次分離。在豬用口服疫苗研究方面,PAdV-3在腸道嗜性、免疫原性和逃逸自身載體免疫方面,顯著優(yōu)于目前普遍使用的人5型腺病毒(HAdV-5)。此外,在人用疫苗研究方面,由于PAdV-3與HAdV-5無血清交叉反應(yīng),避免了人體普遍存在HAdV-5載體免疫的干擾和造成潛在的組織損傷,因此PAdV-3比HAdV-5也更具安全性。充分發(fā)揮PAdV-3以上優(yōu)勢的前提是具備缺失E1區(qū)豬3型腺病毒(PAdV-3ΔE1)的感染性克隆和高效包裝細(xì)胞系。本實(shí)驗(yàn)室目前已擁有PAdV-3ΔE1感染性克隆,但是由于已報道包裝PAdV-3ΔE1的細(xì)胞系VR1BL(穩(wěn)定表達(dá)HAdV-5 E1和PAdV-3 E1Blarge的豬視網(wǎng)膜上皮細(xì)胞系)受到多達(dá)13項專利保護(hù),因此,基于PAdV-3ΔE1的口服疫苗及基因遞送系統(tǒng)研究受到了極大的限制�？紤]到野生型PAdV-3除了可在VR1BL中增殖外,還可在豬睪丸(swine testicular,ST)細(xì)胞中高效增殖,本研究基于第二代重組慢病毒包裝系統(tǒng),構(gòu)建可穩(wěn)定表達(dá)PAdV-3 E1Blarge和I-SceI的細(xì)胞系——ST-E1-E1Blarge-ISceI。與專利保護(hù)的VR1BL細(xì)胞相比,ST-E1-E1Blarge-ISce I細(xì)胞可實(shí)現(xiàn)PAdV-3ΔE1的成功包裝和高效增殖,病毒包裝能力略低于VR1BL細(xì)胞,但96 h內(nèi)病毒增殖能力比VR1BL細(xì)胞高100倍。本研究的主要內(nèi)容和結(jié)果如下:1.第二代重組慢病毒的包裝構(gòu)建第二代慢病毒包裝用克隆載體。在本實(shí)驗(yàn)室已構(gòu)建的克隆載體的基礎(chǔ)上,合成連接肽3HA-MCS1-2A-MCS2,將合成片段連接到克隆載體上構(gòu)建成pTrip-CMV-3HA-MCS1-2A-MCS2-IRES-Hygro。在MSC1處插入3NLS-ISceI基因,在MSC2處插入His-E1Blarge基因序列。PCR、酶切鑒定及測序正確的載體,按照操作步驟將包裝載體psPAX2、病毒衣殼載體pMD2.G、克隆載體共轉(zhuǎn)染293T細(xì)胞,包裝慢病毒后測定病毒滴度。進(jìn)而從慢病毒抗性篩選和蛋白表達(dá)兩個方面驗(yàn)證重組慢病毒包裝成功。2.ST-E1-E1Blarge-ISceI細(xì)胞系的篩選10 MOI重組慢病毒轉(zhuǎn)導(dǎo)ST-E1細(xì)胞,按照標(biāo)準(zhǔn)操作步驟進(jìn)行潮霉素篩選細(xì)胞,連續(xù)篩選3輪后,挑取細(xì)胞單克隆傳代培養(yǎng),分別收集第5代、第10代和第30代細(xì)胞樣品,用Western blot方法檢測細(xì)胞中是否穩(wěn)定表達(dá)PAdV-3 E1Blarge和I-SceI,分別用抗HA鼠單抗和抗His鼠單抗作為一抗。實(shí)驗(yàn)結(jié)果表明,ST-E1-E1Blarge-ISceI細(xì)胞篩選成功并能穩(wěn)定表達(dá)PAdV-3 E1Blarge和I-SceI。3.檢測PAdV-3ΔE1在VR1BL和ST-E1-E1Blarge-ISceI上拯救和增殖能力將本實(shí)驗(yàn)室已有的PAd V-3ΔE1基因組載體,經(jīng)Pac I酶切線性化,使用Lipofectin reagent轉(zhuǎn)染VR1BL和ST-E1-E1Blarge-ISceI細(xì)胞,包裝E1區(qū)表達(dá)綠色熒光的PAdV-3病毒(PAdV219),觀察轉(zhuǎn)染效率和測定包裝病毒的滴度。實(shí)驗(yàn)結(jié)果表明,本實(shí)驗(yàn)室篩選的細(xì)胞可以包裝PAd V-3ΔE1,但病毒基因組轉(zhuǎn)染效率和病毒包裝效率略低于VR1BL細(xì)胞。在病毒增殖能力方面,72 h內(nèi)本文構(gòu)建細(xì)胞與VR1BL細(xì)胞具有相似增殖能力。但在72-96 h之間,由于本文構(gòu)建細(xì)胞存活率仍接近50%,而VR1BL細(xì)胞全部脫落死亡,所以本文構(gòu)建細(xì)胞病毒增殖滴度比VR1BL細(xì)胞高出100倍。綜合以上實(shí)驗(yàn)結(jié)果表明,本實(shí)驗(yàn)室篩選細(xì)胞系具有高效PAdV-3ΔE1包裝和增殖能力,并有望極大促進(jìn)基于PAdV-3ΔE1口服載體疫苗和基因遞送系統(tǒng)的研究。
[Abstract]:Porcine adenovirus type 3 (PAdV-3) in 1964 for the first time isolated from healthy pigs. The pigs in the intestinal tract of the oral vaccine, eosinophilic intestinal PAdV-3, immunogenicity and immune escape its carrier, significantly better than that of the human adenovirus type 5 (HAdV-5). In addition, people used in vaccine research no, because the serum cross reaction of PAdV-3 and HAdV-5, to avoid the human universal vector of HAdV-5 interference and immune injury potential, so PAdV-3 is more than HAdV-5. The premise of safety and give full play to PAdV-3 advantage is above E1 deletion region of porcine adenovirus type 3 (PAdV-3 E1) and the infectious clone efficient packaging cell line. The laboratory now has PAdV-3 E1 infectious clone, but because VR1BL cells have been reported PAdV-3 packaging E1 Delta (stable pig retinal pigment epithelial cell line expressing HAdV-5 E1 and PAdV-3 E1Blarge) by up to 1 3 items of patent protection, therefore, oral vaccine and gene delivery system of PAdV-3 E1 has been greatly restricted. Based on the consideration of wild type PAdV-3 in addition to the proliferation in VR1BL, but also in the pig testis (swine testicular, ST), the proliferation of cells, second generation recombinant lentiviral packaging system based on this research construction of ST-E1-E1Blarge-ISceI. cells, and the patent protection system with stable expression of PAdV-3 E1Blarge and I-SceI VR1BL cells compared to I cells, ST-E1-E1Blarge-ISce can achieve the success of the E1 PAdV-3 delta packaging and high proliferation, virus packaging capacity is slightly lower than that of VR1BL cells, but the 96 h virus proliferation of VR1BL cells was 100 times higher than the main content and results of this. The research is as follows: 1. the second generation of recombinant lentivirus packaging construction of the second generation lentivirus packaging cloning vector. Based on cloning vectors have been constructed in our lab on a connection The synthetic peptide 3HA-MCS1-2A-MCS2, cloned into cloning vector to construct pTrip-CMV-3HA-MCS1-2A-MCS2-IRES-Hygro. 3NLS-ISceI gene in MSC1, insert the His-E1Blarge.PCR gene sequence in MSC2, enzyme digestion identification and sequencing vector, according to the operation steps of loading package psPAX2, the viral capsid pMD2.G vector cloning vector were transfected into 293T cells. The viral titer was determined by packaging slow virus. And then screened from slow virus resistance and protein expression in two aspects to verify the recombinant lentiviral packaging cell line.2.ST-E1-E1Blarge-ISceI successfully screened 10 MOI recombinant lentiviral transduction of ST-E1 cells, in accordance with the standard operating procedure of hygromycin screening cells, continuous screening after the 3 round, picked the cell monoclonal passaging, were collected from fifth generation. The tenth and thirtieth generation cell samples, using Western blot method to detect whether cells with stable expression of PAdV-3 E1Bla Rge and I-SceI, respectively, with anti HA monoclonal antibody and anti mouse His monoclonal antibody as the first antibody. The experimental results show that ST-E1-E1Blarge-ISceI cells successfully screened and stably expressed PAdV-3 E1Blarge and I-SceI.3. PAdV-3 detection in VR1BL and ST-E1-E1Blarge-ISceI E1 to save on the proliferation and PAd V-3 E1 genome vector of this laboratory has, by Pac I were linearized with Lipofectin reagent transfected VR1BL and ST-E1-E1Blarge-ISceI cells, green fluorescent virus expressing PAdV-3 E1 region (PAdV219), packaging and packaging to observe the transfection efficiency determination of virus titer. The experimental results show that the laboratory screening cells can PAd V-3 a E1 package, but the transfection efficiency of virus genome and virus packaging efficiency slightly lower than that of VR1BL cells. The virus proliferation, we construct 72 h cells and VR1BL cells have similar proliferation ability. But in the 72-96 h, because this Construction of cell survival rate is close to 50%, while the VR1BL cell death all off, so the construction of cell proliferation of virus titer is 100 times higher than that of VR1BL cells. The above results show that the laboratory screening of cell lines with high PAdV-3 E1 packaging and proliferation, and is expected to greatly promote the research of PAdV-3 E1 oral vaccine and gene carrier based on the delivery system.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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