本文選題：ASP-1　切入點(diǎn)：佐劑　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文

【摘要】： 本文研究的活化相關(guān)分泌蛋白-1(ASP-1, activation associated secreted protein-1)是一種來源于盤尾絲蟲的蛋白,是一種非毒素類微生物衍生物型佐劑。目前已報(bào)道的初步實(shí)驗(yàn)研究顯示,其具有良好的佐劑活性。本課題以ASP-1蛋白為研究對象,研究了其在原核系統(tǒng)表達(dá)的佐劑效應(yīng)及特征,并對ASP-1蛋白在酵母系統(tǒng)中的表達(dá)及佐劑功能進(jìn)行了初步的研究。 一、ASP-1蛋白在原核系統(tǒng)中的表達(dá)及其佐劑活性分析 1、ASP-1基因的合成、克隆及序列分析 根據(jù)Genebank登錄的ASP-1序列,使用Upgene軟件設(shè)計(jì)了36條引物(正反向各18條),采用重疊PCR方法,通過兩輪PCR擴(kuò)增,合成了ASP-1基因。產(chǎn)物克隆至T載體,雙酶切后連接到pQE30載體中。經(jīng)序列測定成功獲得經(jīng)過大腸桿菌密碼子優(yōu)化的目的基因。 2、ASP-1在原核表達(dá)系統(tǒng)中的表達(dá)、純化和復(fù)性 ASP-1蛋白在原核大腸桿菌中以難溶的包涵體形式大量表達(dá)。經(jīng)SDS-PAGE, Western-blot方法鑒定正確后,進(jìn)一步將重組蛋白ASP-1通過鎳離子柱進(jìn)行親和層析純化,獲得了高純度的ASP-1蛋白。最終將純化的ASP-1蛋白通過梯度透析的方法得到復(fù)性的可溶性重組蛋白。 3、以O(shè)VA模式抗原分析ASP-1的佐劑活性 在對常用模式抗原卵清蛋白OVA免疫實(shí)驗(yàn)中,我們發(fā)現(xiàn)ASP-1佐劑組在誘導(dǎo)細(xì)胞和體液免疫應(yīng)答水平均明顯高于鋁佐劑組和無佐劑對照組,尤其是相比較鋁佐劑組在細(xì)胞免疫水平上對比更加明顯。雖然OVA抗原最終仍是以Th2型免疫應(yīng)答為主(IgG2aIgG1),但Th1型應(yīng)答所占的比重明顯升高,說明ASP-1表現(xiàn)出明顯的同時(shí)誘導(dǎo)細(xì)胞與體液免疫應(yīng)答的能力。 4、ASP-1蛋白在滅活疫苗聯(lián)合免疫中的佐劑效應(yīng)研究 我們選擇了三種常用滅活病毒疫苗:流行性出血熱,流感和狂犬疫苗。結(jié)果表明,三聯(lián)疫苗免疫時(shí),不加ASP-1蛋白,IgG1/IgG2a滴度均不及或相當(dāng)于各自單獨(dú)免疫時(shí)水平;而加入ASP-1蛋白佐劑后,盡管各自用量減半,獲得的免疫效果不論是Th1還是Th2應(yīng)答方面甚至超過各自單獨(dú)免疫的水平。說明了ASP-1佐劑的加入,可以達(dá)到減少疫苗各自的用量,誘導(dǎo)平衡Th1/Th2應(yīng)答的目的。 二、ASP-1在畢氏酵母系統(tǒng)中的表達(dá)及其佐劑活性分析 在原核大腸桿菌中表達(dá)重組ASP-1蛋白的動(dòng)物實(shí)驗(yàn)中,已經(jīng)表明其具有良好的佐劑活性,但原核表達(dá)的包涵體難以溶解且復(fù)性過程煩瑣,還可能喪失天然蛋白的某些活性。所以為了獲得更加接近于天然結(jié)構(gòu)的可溶性蛋白,我們采用了巴斯德畢氏酵母表達(dá)系統(tǒng)進(jìn)行表達(dá)。 首先利用Vector NTI軟件設(shè)計(jì)出兩條引物,將PCR擴(kuò)增產(chǎn)物連接到T載體,通過雙酶切的方法將目的片斷ASP-1克隆到pPic9k載體中,PCR方法鑒定陽性克隆。隨后將轉(zhuǎn)化子轉(zhuǎn)接到含有不同抗性濃度的G418培養(yǎng)基和MM、MD平板中篩選高拷貝株和不同的誘導(dǎo)表型。 本實(shí)驗(yàn)酵母表達(dá)選擇甲醇利用緩慢的Muts型來進(jìn)行誘導(dǎo)表達(dá)。ASP-1蛋白在酵母中的表達(dá)分為兩步,即菌體生長和蛋白誘導(dǎo)。先在以甘油為碳源的培養(yǎng)基上培養(yǎng),當(dāng)菌液OD值達(dá)到2-5后,將菌體懸浮于以甲醇為碳源的培養(yǎng)基中誘導(dǎo)表達(dá)。每隔24小時(shí)補(bǔ)加一次甲醇,分別在不同的時(shí)間段收集上清取樣。樣品最后經(jīng)過SDS-PAGE, ELISA, Western-blot等方法進(jìn)行鑒定。 在用酵母表達(dá)的ASP-1蛋白進(jìn)行功能性研究的過程中,我們同樣選擇了模式抗原OVA和狂犬滅活疫苗進(jìn)行分析。初步結(jié)果顯示,在與狂犬疫苗和OVA共同免疫BALB/c小鼠時(shí),重組蛋白ASP-1表現(xiàn)出了較強(qiáng)的同時(shí)誘導(dǎo)細(xì)胞與體液免疫應(yīng)答的佐劑活性。 通過以上的研究說明,ASP-1蛋白具有良好的佐劑效應(yīng),有望發(fā)展成為一種新型的人用佐劑,值得深入研究。
[Abstract]:This paper studies the activation of secretory proteins associated with -1 (ASP-1, activation associated secreted protein-1) is a protein derived from Onchocerca volvulus, is a non toxin derivative microbial adjuvant. Preliminary experimental study has been reported that it has adjuvant activity. The ASP-1 protein as the research object. Study on the adjuvant effect and characteristics of prokaryotic expression system, and the expression of ASP-1 protein in yeast and adjuvant system were studied.
Expression of ASP-1 protein in the prokaryotic system and analysis of the activity of its adjuvant
1, ASP-1 gene synthesis, cloning and sequence analysis
According to the sequence of ASP-1 Genebank login, 36 primers were designed using Upgene software (positive and negative 18), using overlapping PCR method, through two rounds of PCR amplification, ASP-1 gene was synthesized. The products were cloned into T vector. After digested and ligated to pQE30 vector. After sequencing the success through the password. Optimization of Escherichia gene.
2, the expression, purification and renaturation of ASP-1 in the prokaryotic expression system
The expression of ASP-1 protein in prokaryotic Escherichia coli in inclusion insoluble form. By SDS-PAGE Western-blot method, after correct identification, the recombinant ASP-1 protein was purified by nickel ion affinity chromatography column, the purity of the ASP-1 protein. Finally the purified ASP-1 protein by gradient dialysis refolding obtained the soluble recombinant protein.
3, analysis of the adjuvant activity of ASP-1 by OVA model antigen
In the common mode OVA antigen ovalbumin immune experiment, we found that ASP-1 adjuvant group induced cellular and humoral immune responses were distinctly higher than that of aluminum adjuvant group and adjuvant control group, especially compared with aluminum adjuvant group in the cellular immunity level in contrast to the more obvious. Although OVA is still the final antigen with Th2 type immune response (mainly IgG2aIgG1), but the Th1 type response proportion increased significantly, indicating that ASP-1 also showed obvious ability to induce cellular and humoral immune responses.
Study on the adjuvant effect of 4, ASP-1 protein in inactivated vaccine combined immunization
We selected three kinds of inactivated virus vaccine, epidemic hemorrhagic fever, influenza and rabies vaccine. The results show that the triple vaccine, with ASP-1 protein, IgG1/IgG2a titers were less than or equal to separate immunization level; and ASP-1 protein added adjuvant, even though each half of the amount, whether it is the immune effect Th1 or Th2 response even more than their individual immune level. The addition of ASP-1 adjuvant, can reduce the dosage of vaccine induced Th1/Th2 response, balance.
Expression of two, ASP-1 in Pichia yeast system and analysis of the activity of its adjuvant
Animal experiment of recombinant ASP-1 protein expressed in prokaryotic Escherichia coli, had shown that it has good adjuvant activity, but the inclusion of prokaryotic expression and renaturation process cumbersome and difficult to dissolve, some activity may also lose the natural protein. So in order to get closer to the natural protein structure, we use the Pasteur Pichia expression system for expression.
We use Vector NTI software to design two primers, PCR amplification products connected to the T vector method by double enzyme digestion to fragment ASP-1 was cloned into pPic9k vector and the PCR method. The positive clones were identified and then transferred to the transformants with different resistance concentration of G418 medium and MM, MD in flat screen high copy lines and different induced phenotype.
The yeast expression of type Muts selection of methanol utilization slow to induce the expression of.ASP-1 protein expression in yeast is divided into two steps, namely, cell growth and protein induction. First cultured in medium with glycerol as the carbon source, when the OD value reaches 2-5, the cells were suspended in methanol to induce the expression of carbon source in the medium. Every 24 hours feeding time of methanol, the supernatant samples at different time respectively. Finally the sample after SDS-PAGE, ELISA, Western-blot and other methods were identified.
The process of functional studies in yeast ASP-1 protein expression, we also choose the mode of antigen OVA and inactivated rabies vaccine were analyzed. Preliminary results show that with rabies vaccine and OVA immunization of BALB/c mice, the recombinant ASP-1 protein showed strong activity with adjuvant induced cellular and humoral immune response.
According to the above studies, ASP-1 protein has a good adjuvant effect and is expected to develop into a new type of human adjuvant, which is worthy of further study.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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