轮状病毒结构蛋白VP4在家蚕杆状病毒表达系统中的表达
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摘要
轮状病毒(rotavirus,RV)是引起全球婴幼儿及幼畜非细菌性胃肠炎的主要病原体之一,VP4蛋白是轮状病毒的主要中和抗原。利用载体RD-BmBacmid与重组载体pBacPAK8-VP4共转染家蚕细胞BmN获得重组家蚕杆状病毒BmNPV-VP4,并用该病毒感染BmN细胞及家蚕5龄幼虫,从而在BmN细胞及家蚕中表达TB-Chen株轮状病毒的VP4蛋白。Western blotting检测到BmN细胞及家蚕幼虫血淋巴中均出现88 kD的特异性条带,证明VP4蛋白在BmN细胞和家蚕中获得了表达。ELISA检测结果显示,VP4蛋白在BmN细胞和家蚕幼虫血淋巴中的表达分别在感染后4 d和6 d达到最高水平,每个细胞和每mL血淋巴中的表达量分别为4.28 pg和1.61μg。研究结果为轮状病毒口服疫苗的研制奠定了基础。
Rotavirus(RV) is one of the major pathogens causing global non-bacterial gastroenteritis in infants and young animals. VP4 protein is the main neutralizing antigen of rotavirus. In this experiment, BmN cells were transfected with RD-BmBacmid and recombinant vector pBacPAK8-VP4 to obtain recombinant baculovirus BmNPV-VP4. Then the virus was used to inoculate BmN cells and silkworm larvae of 5 th instar so that VP4 protein of TB-Chen rotavirus could be expressed in BmN cells and silkworm. Western blotting analysis indicated that there were specific bands of 88 kD in both samples from BmN cells and hemolymph of silkworm larvae, which proved that this protein was expressed in BmN cells and silkworm. ELISA analysis showed that the expression of VP4 protein in BmN cells reached the maximum value of 4.28 pg/cell on the 4 th day, and the expression of VP4 protein in hemolymph of silkworm larvae reached the maximum level of 1.61 μg/mL on the 6 th day after infection. This study laid a foundation for the development of oral vaccine against rotavirus.
Rotavirus(RV) is one of the major pathogens causing global non-bacterial gastroenteritis in infants and young animals. VP4 protein is the main neutralizing antigen of rotavirus. In this experiment, BmN cells were transfected with RD-BmBacmid and recombinant vector pBacPAK8-VP4 to obtain recombinant baculovirus BmNPV-VP4. Then the virus was used to inoculate BmN cells and silkworm larvae of 5 th instar so that VP4 protein of TB-Chen rotavirus could be expressed in BmN cells and silkworm. Western blotting analysis indicated that there were specific bands of 88 kD in both samples from BmN cells and hemolymph of silkworm larvae, which proved that this protein was expressed in BmN cells and silkworm. ELISA analysis showed that the expression of VP4 protein in BmN cells reached the maximum value of 4.28 pg/cell on the 4 th day, and the expression of VP4 protein in hemolymph of silkworm larvae reached the maximum level of 1.61 μg/mL on the 6 th day after infection. This study laid a foundation for the development of oral vaccine against rotavirus.
引文
[1] 汪洋,白萱,余晖,等.2006年武汉地区A组轮状病毒VP7基因型初步研究[J].黄冈师范学院学报,2006,26(6):69-72
[2] 张艳,文喻玲,魏海涛,等.A组轮状病毒VP5*和VP8*的表达和免疫学性质研究[J].中国生物工程杂志,2010,30(2):32-38
[3] 黄仕和,余模松.测定杆状病毒滴度方法的研究进展[J].微生物学免疫学进展,2007,35(2):79-83
[4] WU X F,YIN Z Z,CAO C P,et al.Expression of human VEGF165 in silkworm (Bombyx mori L.) by using a recombinant baculovirus and its bioactivity assay[J].J Biotechnol,2004,111(3):253-261
[5] 贾连智,李廷栋,葛胜祥.轮状病毒VP4亚单位疫苗研究进展[J].生物工程学报,2017,33(7):1075-1084
[6] WARD R L,KNOWLTON D R,GREENBERG H B,et al.Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus[J].J Virol,1990,64(6):2687-2691
[7] CHOI A H C,BASU M,MCNAEL M M,et al.Antibody-indepen-dent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein[J].J Virol,1999,73(9):7574-7581
[8] PATEL M,GLASS R I,JIANG B M,et al.A systematic review of anti-rotavirus serum IgA antibody titer as a potential correlate of rotavirus vaccine efficacy[J].J Infect Dis,2013,208(2):284-294
[9] GONG Z,JIN Y,ZHANG Y.Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes[J].J Biotechnol,2005,119(1):93-105
[10] 费伟强,陈琴,陈倩,等.复制缺陷型BmNPV载体的构建及初步应用[J].蚕业科学,2013,39(3):514-521
[11] BRAKE A J,MERRYWEATHER J P,COIT D G,et al.α-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae[J].Proc Natl Acad Sci USA,1984,81(15):4642-4646
[12] 王宝贵,孙晓霞,赵林伊,等.柞蚕蛹蛋白质营养价值研究[J].中国公共卫生,2002,18(5):67-68
[13] 杜仲争,庄大桓.全蚕粉及其生理功能[J].中国蚕业,2000,22(2):53-54
[14] 桂仲争,庄大桓,戴建一,等.资源昆虫(家蚕)的食用与药用价值研究进展[J].中国蚕业,2002,24(4):63-65
[15] CHEN J,WU X F,ZHANG Y Z.Expression,purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor[J].J Biotechnol,2006,123(2):236-247
[16] ZHANG W P,WANG J,ZHANG C,et al.Synergetic protein factors that improve rhGM-CSF absorption via an oral route exist in silkworm pupae[J].Mol Pharm,2015,12(5):1347-1355
[2] 张艳,文喻玲,魏海涛,等.A组轮状病毒VP5*和VP8*的表达和免疫学性质研究[J].中国生物工程杂志,2010,30(2):32-38
[3] 黄仕和,余模松.测定杆状病毒滴度方法的研究进展[J].微生物学免疫学进展,2007,35(2):79-83
[4] WU X F,YIN Z Z,CAO C P,et al.Expression of human VEGF165 in silkworm (Bombyx mori L.) by using a recombinant baculovirus and its bioactivity assay[J].J Biotechnol,2004,111(3):253-261
[5] 贾连智,李廷栋,葛胜祥.轮状病毒VP4亚单位疫苗研究进展[J].生物工程学报,2017,33(7):1075-1084
[6] WARD R L,KNOWLTON D R,GREENBERG H B,et al.Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus[J].J Virol,1990,64(6):2687-2691
[7] CHOI A H C,BASU M,MCNAEL M M,et al.Antibody-indepen-dent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein[J].J Virol,1999,73(9):7574-7581
[8] PATEL M,GLASS R I,JIANG B M,et al.A systematic review of anti-rotavirus serum IgA antibody titer as a potential correlate of rotavirus vaccine efficacy[J].J Infect Dis,2013,208(2):284-294
[9] GONG Z,JIN Y,ZHANG Y.Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes[J].J Biotechnol,2005,119(1):93-105
[10] 费伟强,陈琴,陈倩,等.复制缺陷型BmNPV载体的构建及初步应用[J].蚕业科学,2013,39(3):514-521
[11] BRAKE A J,MERRYWEATHER J P,COIT D G,et al.α-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae[J].Proc Natl Acad Sci USA,1984,81(15):4642-4646
[12] 王宝贵,孙晓霞,赵林伊,等.柞蚕蛹蛋白质营养价值研究[J].中国公共卫生,2002,18(5):67-68
[13] 杜仲争,庄大桓.全蚕粉及其生理功能[J].中国蚕业,2000,22(2):53-54
[14] 桂仲争,庄大桓,戴建一,等.资源昆虫(家蚕)的食用与药用价值研究进展[J].中国蚕业,2002,24(4):63-65
[15] CHEN J,WU X F,ZHANG Y Z.Expression,purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor[J].J Biotechnol,2006,123(2):236-247
[16] ZHANG W P,WANG J,ZHANG C,et al.Synergetic protein factors that improve rhGM-CSF absorption via an oral route exist in silkworm pupae[J].Mol Pharm,2015,12(5):1347-1355