乙型肝炎新型植物口服疫苗的研究
發(fā)布時間：2018-09-09 16:14
【摘要】： 乙型肝炎(Hepatitis B,HB)是一種由乙型肝炎病毒(Hepatitis B virus,HBV)感染所引起的傳染性疾病,我國大約有10%的人群是乙肝病毒攜帶者。目前仍然缺乏有效的治療手段治療HB,因此,主要靠接種疫苗來進行預防HB。由于現在使用商業(yè)化HBV疫苗主要通過酵母表達系統(tǒng)生產,存在較高的生產成本,而且需要冷藏,因此對邊遠和經濟不發(fā)達地區(qū)人群疫苗接種普及帶來困難。此外,大約有10%的接種人群對現在商業(yè)化HB疫苗不產生明顯免疫反應。因此,探索研制新型價廉的HB疫苗成為疫苗研究關注的焦點。本研究在國內外已有工作基礎上,對人新型HBV表面抗原融合基因SS1進行了改造,并將SS1基因轉入植物中,開展了相關研究,取得以下主要結果。 1、為了分析新型乙肝表面抗原SS1(在乙肝主要表面抗原(S)的C端融合了乙肝表面抗原前體肽preS1(21-47aa))是否具有preS1的免疫原性,并在體內激起針對preS1的抗體反應,我們依照大腸桿菌B株系的密碼子偏愛特性改造和合成了全長的adr血清型preS1基因(spreS1)并在大腸桿菌進行了表達。結果顯示表達產物以可溶性形式存在,表達量占到總蛋白的44%,經過Ni-NTA親和層析純化,純化的蛋白純度達到98%以上,western blot和間接ELISA結果表明該重組蛋白具有preS1的抗原性,而且抗原性與現在商業(yè)化的preS1相當。這些克服了以往在表達全長preS1蛋白時遇到的包涵體、表達產物不穩(wěn)定或低表達的難題。 2、為了探索生產一種新型的HB口服疫苗形式,我們選擇了水稻作為新型乙肝表面抗原SS1的表達宿主。我們通過目前的文獻報道選取了水稻種子表達的高效表達啟動子P_(GluB-4),構建乙型肝炎抗原基因SS1的水稻種子高效表達載體p1300GSS1,通過農桿菌轉化,實現了其在水稻種子中的表達。重組蛋白的表達量為31.5 ng/g DW種子,且能夠自組裝成病毒樣顆粒結構(virus-like particles,VLP),顆粒的粒徑大約為22±2 nm,沉降系數為1.25 g.cm~(-3)。Western blot結果顯示同時具有S和preS1抗原性。動物免疫試驗顯示重組蛋白能夠在小鼠體內激起針對S和preS1的抗體反應,這些為今后的臨床評價提供理論基礎。 3、為了進一步提高新型乙肝表面抗原SS1在植物體內的表達水平,我們首次嘗試利用植物葉綠體表達系統(tǒng)進行這種新型乙肝表面抗原的表達。越來越多的研究表明植物葉綠體表達系統(tǒng)具有諸多優(yōu)越性,其中外源基因的高效表達最引人注目。乙肝表面抗原是一種糖蛋白,而且它的糖基化程度與其抗原性直接相關,本研究構建了其煙草的葉綠體表達載體并進行了煙草的遺傳轉化,結果顯示外源基因被正確地整合到葉綠體基因組中預定的位點,表達的重組蛋白經乙肝表面抗原檢測試劑盒檢測具有抗原性,相關的研究還在繼續(xù)中。 4、為了提高口服疫苗的免疫效果,我們對目前常用的免疫佐劑分子霍亂毒素B亞基蛋白(cholera toxin B,CTB)進行了大腸桿菌表達。本研究首先克隆了去除信號肽編碼序列的霍亂毒素B亞基基因CTB,構建了大腸桿菌表達載體,進行表達,結果顯示表達產物以包函體的形式出現,表達量占到總蛋白的25%,經過包函體的變性和復性,純化的蛋白純度達到98%,體外生活性試驗表明純化的蛋白主要以五聚體的形式出現,western blot結果顯示五聚體大約占到總純化蛋白的66.8%。由于現在商業(yè)化的疫苗中還沒有添加任何佐劑成份,尤其是CTB蛋白,所以口服免疫佐劑表達系統(tǒng)的建立為今后水稻表達的新型乙肝表面抗原的動物口服試驗提供了基礎。
[Abstract]:Hepatitis B (HB) is an infectious disease caused by hepatitis B virus (HBV) infection. About 10% of the population in China is hepatitis B virus carriers. There is still a lack of effective treatment for HB, so vaccination is the main way to prevent HB. It is difficult to vaccinate people in remote and economically underdeveloped areas because of the high production cost and the need for refrigeration. In addition, about 10% of the vaccinated population do not have obvious immune response to the commercial HB vaccine. Therefore, exploring new and inexpensive HB vaccine becomes epidemic. In this study, a novel human HBV surface antigen fusion gene SS1 was modified and transformed into plants based on the previous work at home and abroad.
1. In order to analyze the immunogenicity of a novel hepatitis B surface antigen SS1 (preS1 (21-47aa), fused with a hepatitis B surface antigen precursor peptide preS1 (21-47aa) at the C terminal of the main hepatitis B surface antigen (S), and to stimulate antibody response against preS1 in vivo, we modified and synthesized a full-length ADR serum according to the codon preference characteristics of E. coli B strain. PreS1 gene (spreS1) was expressed in E. coli. The results showed that the expressed product was soluble and accounted for 44% of the total protein. The purity of the purified protein was over 98% after purification by Ni-NTA affinity chromatography. Western blot and indirect ELISA showed that the recombinant protein was antigenic and antigenic to preS1. The commercialized preS1 is comparable now. These overcome the difficulties encountered in expressing full-length preS1 proteins, including inclusion bodies, unstable or low expression products.
2. In order to explore a new form of oral HB vaccine, we chose rice as the expression host of a novel hepatitis B surface antigen SS1. The recombinant protein was expressed in rice seeds by Agrobacterium tumefaciens transformation. The expression of the recombinant protein was 31.5 ng/g DW, and it could self-assemble into virus-like particles (VLP). The particle size was about 22 2 nm, and the sedimentation coefficient was 1.25 g.cm~(-3). Western blot results showed that the recombinant protein had both S and preS1 antigenicity. Immunoassay showed that the recombinant protein could stimulate antibody response against S and preS1 in mice, which provided theoretical basis for future clinical evaluation.
3. In order to further improve the expression level of the novel hepatitis B surface antigen SS1 in plants, we first attempted to use the plant chloroplast expression system to express this novel hepatitis B surface antigen. Objective. Hepatitis B surface antigen is a glycoprotein, and its glycosylation degree is directly related to its antigenicity. In this study, we constructed its tobacco chloroplast expression vector and transformed it into tobacco. The results showed that the exogenous genes were correctly integrated into the predetermined sites in the chloroplast genome, and the recombinant protein was expressed on the surface of hepatitis B. Antigen detection kit has antigenicity, and related research is continuing.
4. In order to improve the immune effect of oral vaccine, cholera toxin B subunit protein (CTB) was expressed in E. coli. In this study, the cholera toxin B subunit gene CTB was cloned to remove the coding sequence of signal peptide, and the expression vector of E. coli was constructed. The purity of the purified protein was 98% after denaturation and renaturation of the inclusion bodies. In vitro bioassay showed that the purified protein was mainly in the form of pentamer. Western blot showed that pentamer accounted for about 66.8% of the total purified protein. No adjuvant, especially CTB protein, has been added to commercial vaccines, so the establishment of an oral immune adjuvant expression system will provide a basis for future animal oral trials of novel hepatitis B surface antigen expressed in rice.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392
本文編號：2232897
[Abstract]:Hepatitis B (HB) is an infectious disease caused by hepatitis B virus (HBV) infection. About 10% of the population in China is hepatitis B virus carriers. There is still a lack of effective treatment for HB, so vaccination is the main way to prevent HB. It is difficult to vaccinate people in remote and economically underdeveloped areas because of the high production cost and the need for refrigeration. In addition, about 10% of the vaccinated population do not have obvious immune response to the commercial HB vaccine. Therefore, exploring new and inexpensive HB vaccine becomes epidemic. In this study, a novel human HBV surface antigen fusion gene SS1 was modified and transformed into plants based on the previous work at home and abroad.
1. In order to analyze the immunogenicity of a novel hepatitis B surface antigen SS1 (preS1 (21-47aa), fused with a hepatitis B surface antigen precursor peptide preS1 (21-47aa) at the C terminal of the main hepatitis B surface antigen (S), and to stimulate antibody response against preS1 in vivo, we modified and synthesized a full-length ADR serum according to the codon preference characteristics of E. coli B strain. PreS1 gene (spreS1) was expressed in E. coli. The results showed that the expressed product was soluble and accounted for 44% of the total protein. The purity of the purified protein was over 98% after purification by Ni-NTA affinity chromatography. Western blot and indirect ELISA showed that the recombinant protein was antigenic and antigenic to preS1. The commercialized preS1 is comparable now. These overcome the difficulties encountered in expressing full-length preS1 proteins, including inclusion bodies, unstable or low expression products.
2. In order to explore a new form of oral HB vaccine, we chose rice as the expression host of a novel hepatitis B surface antigen SS1. The recombinant protein was expressed in rice seeds by Agrobacterium tumefaciens transformation. The expression of the recombinant protein was 31.5 ng/g DW, and it could self-assemble into virus-like particles (VLP). The particle size was about 22 2 nm, and the sedimentation coefficient was 1.25 g.cm~(-3). Western blot results showed that the recombinant protein had both S and preS1 antigenicity. Immunoassay showed that the recombinant protein could stimulate antibody response against S and preS1 in mice, which provided theoretical basis for future clinical evaluation.
3. In order to further improve the expression level of the novel hepatitis B surface antigen SS1 in plants, we first attempted to use the plant chloroplast expression system to express this novel hepatitis B surface antigen. Objective. Hepatitis B surface antigen is a glycoprotein, and its glycosylation degree is directly related to its antigenicity. In this study, we constructed its tobacco chloroplast expression vector and transformed it into tobacco. The results showed that the exogenous genes were correctly integrated into the predetermined sites in the chloroplast genome, and the recombinant protein was expressed on the surface of hepatitis B. Antigen detection kit has antigenicity, and related research is continuing.
4. In order to improve the immune effect of oral vaccine, cholera toxin B subunit protein (CTB) was expressed in E. coli. In this study, the cholera toxin B subunit gene CTB was cloned to remove the coding sequence of signal peptide, and the expression vector of E. coli was constructed. The purity of the purified protein was 98% after denaturation and renaturation of the inclusion bodies. In vitro bioassay showed that the purified protein was mainly in the form of pentamer. Western blot showed that pentamer accounted for about 66.8% of the total purified protein. No adjuvant, especially CTB protein, has been added to commercial vaccines, so the establishment of an oral immune adjuvant expression system will provide a basis for future animal oral trials of novel hepatitis B surface antigen expressed in rice.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392
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