【作者】 曹轶梅；

【导师】 刘在新；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2009， 博士

【摘要】 2005年我国江苏等省暴发Asia 1型口蹄疫(foot-and-mouth disease, FMD),对国民经济造成巨大损失,引起此次流行的口蹄疫病毒(foot-and-mouth disease, FMDV)与Asia 1/YNBS/1958等历史毒株不同,为新传入的毒株,命名为Asia 1/JS/2005。该毒株除使牛、羊发病外,猪也有确诊病例,说明该毒株对猪也存在很大威胁。本研究以该新发毒株为材料,利用昆虫杆状病毒表达系统,研究Asia 1型FMDV空衣壳的组装,以期研制一种安全、高效的Asia 1型FMDV基因工程空衣壳亚单位疫苗。通过克隆分析细胞传代的Asia 1/JS/05株FMDV的衣壳蛋白P12A和蛋白酶3C基因,表明其P1结构蛋白编码区全长为2145bp,编码715aa。4个结构蛋白VP4、VP2、VP3、VP1分别为207bp、648bp、657bp、633bp,推导的氨基酸序列分别为69aa、216aa、219aa、211aa。各结构蛋白之间的连接氨基酸分别为Ala/Asp(VP4/VP2)、Glu/Gly(VP2/VP3)、Gln/Thr(VP3/VP1)。细胞传代毒的细胞受体结合位点(VP1 143-145)RGD变为RDD。VP3上140位和143位为组氨酸。2A全长48bp,编码16aa。3C全长639bp,编码213aa。分析P12A和3C基因序列与已发表Asia 1/JS/05毒株相应基因序列(GenBank No. EF149009)的核苷酸同源性分别为99.27%、99.4%,氨基酸序列同源性分别为98.9%、99.1%。采用了三种不同的策略来研究Asia 1型FMDV空衣壳的组装:一是将P12A和3C基因序列分别插入分泌型杆状病毒表达载体pMel-Bac B中,探讨目的基因的分泌表达;二是将空衣壳组装必需的3C基因和P12A基因插入带双启动子的表达载体pFastBac? Dual中,两种基因分别置于不同的启动子控制之下,同时表达P12A和3C蛋白,在昆虫细胞内裂解组装空衣壳粒子;三是通过耐酸性改造,研究提高空衣壳酸稳定性的条件;当pH值低于7时,FMDV二十面体对称的衣壳结构就会裂解成12S的五聚体,而昆虫细胞培养基的正常pH值在6.2左右,为了提高空衣壳的耐酸性,应用定点突变技术改变VP3上的H140和H143为亮氨酸,再将改造后的基因插入双启动子载体,以期提高其空衣壳的稳定性。结果表明,(1)采用分泌表达的策略,仅有少量的P12A结构蛋白和3C蛋白分泌到细胞培养基中,但大部分的蛋白仍位于细胞膜的内侧面,没有分泌出来,说明这一策略具有继续完善的空间,需要在基因表达盒的构建和诱导分泌表达的条件方面进行深入的研究。(2)采用双启动子表达自然蛋白的策略,目的蛋白在High Five?细胞中均得到了较高水平的表达, Western blotting分析结果表明衣壳蛋白P12A被3C蛋白酶成功的裂解为VP0、VP1和VP3。免疫电镜观察,在昆虫细胞中组装形成了直径约为25~30nm左右的空衣壳结构,以及直径约为10nm的五聚体结构。将这种粗纯的表达产物(约含1.2μg空衣壳粒子或中间体)经乳化后肌肉注射免疫豚鼠,空衣壳免疫组豚鼠和灭活苗免疫组豚鼠均产生了较高水平的FMDV特异性中和抗体,而野生型杆状病毒感染细胞蛋白对照没有检测到中和抗体,结果证明,杆状病毒表达组装的空衣壳或中间体具有较好的免疫原性,这为深入研制FMD空衣壳亚单位疫苗奠定了基础。(3)改造的P12A基因和3C基因在昆虫细胞中获得较好的表达,通过Western blotting检测也证明衣壳蛋白被3C蛋白酶成功地加工裂解,通过电镜也观察到在昆虫细胞内产生了直径为25～30 nm的空衣壳结构。通过耐酸性分析表明将VP3 H140和H143变为亮氨酸提高了已组装空衣壳的稳定性,但是空衣壳的产量却显著下降,这一研究思路仍然具有继续探索的价值。更多还原

【Abstract】 The outbreaks of foot-and-mouth (FMD) serotype Asia 1 in large areas of China had caused great economic losses since it occurred in Jiangsu Province in 2005. The FMD virus (FMDV) strain that cause these outbreaks was a new isolate different from the history strain Asia 1/YNBS/1958 once appeared in Yunnan province. This new isolate was given the name Asia 1/JS/2005. The virus strain mainly affects ruminant animals and cause clinical and subclinical infection in cattle and sheep, however, pig could also be infected and several clinical cases were reported. In this study, we investigated the assembly of Asia 1 foot-and-mouth disease virus empty capsid particles in insect cells using baculovirus expression system based on this new incurred Asia 1 FMDV, and with the hope to achieve a safe, high potency subunit vaccine based on empty capsid particles for Asia 1 FMDV.The P12A and 3C sequences of cell passaged Asia 1/JS/2005 FMDV were amplified and sequenced. The results showed that the whole length of structural protein coding region was 2145 base pairs (bp) and encoded 715 amino acids (aa). The four capsid proteins, VP4, VP2, VP3, and VP1, were 207bp, 648bp, 657bp, 633bp in length, respectively, and the deduced amino acid sequences were 69aa, 216aa, 219aa, and 211aa. Residues at the VP4/VP2, VP2/VP3, and VP3/VP1 cleavage sites were Ala/Asp, Glu/Gly, and Gln/Thr, which could be recognized and processed by 3C proteinase. The RGD motif in VP1 protein of FMDV changed into RDD in the cell passaged Asia 1/JS/2005. Positions 140aa and 143aa in VP3 were histidine residues which were likely involved in acid sensitivity of empty capsid particles. 2A was 48bp in length and the deduced amino acid sequences were about 16aa, and 3C was 639bp in length and the deduced amino acid sequences were about 213aa.Three different strategies were used to investigate the assembly of empty capsid particles of Asia 1 FMDV. (1) Structural protein P12A and proteinase 3C genes were inserted into the baculovirus transfer vector pMelBac B with a melittin secretion signal sequence to test whether the target proteins could be secreted into culture medium, respectively. (2) A recombinant baculovirus that simultaneously expressed the genes for the P12A and 3C proteins from individual promoters was constructed using baculovirus transfer vector pFastBac? Dual to assembly empty capsid particles in insect cells. (3) Study the factors that affect the capsid stability after assembly in insect cell medium with pH value 6.2. It is demonstrated that the icosahedral virus capsid of FMDV dissociates into 12 pentamers at pH below 7. In this study, two histidine residues at positions 140 and 143 in VP3 were changed into leucine by site-directed mutation to improve acid resistance of empty capsid particles.The results showed: (1) Only a small amount of expressed P12A and 3C proteins were secreted into cell supernatant, and lots of them were unable to secrete into the medium and accumulated in the cell, which indicated the existence of rate-limiting steps in the secretion of foreign proteins. Therefore further study was needed to improve the construction of new cDNA cassettes and conditions for secretory expression of foreign proteins in insect cells. (2) The capsid proteins expressed in High Five? insect cells were successfully processed by viral 3C protease, as shown by Western blotting. Both empty capsid particles with a diameter of about 25~30nm and pentamers with a diameter of approximately 10 nm were observed using immunoelectron microscopy. Furthermore, the empty capsid particles or intermediates induced high levels of FMDV-specific antibodies in guinea pigs following immunization, and neutralizing antibodies were induced in the second week after vaccination. These recombinant, non-infectious, FMDV empty capsids are potentially useful for the development of new diagnostic techniques and vaccines. (3) The modified P12A and 3C were successfully expressed in insect cells by Western blotting, and that expressed capsid proteins were also processed by expressed viral 3C proteinase. Furthermore, only empty capsid particles were observed in lystate of High Five? cells infected with recombinant baculoviruses with mutated P12A under electron microscope, and no pentamers were detected, suggesting that replacing the histidines with leucines could, indeed, improve the stability of empty capsid particles under acidic conditions, but somewhat reduced the efficiency of assembly.更多还原