摘要
猪流行性腹泻(Porcine epidemic diarrhea,PED)是猪的一种以严重腹泻、呕吐和脱水为临床特征的高度接触性传染病。此病多发生于冬季12月至来年2月寒冷季节,在夏季也可发生。哺乳仔猪、断奶仔猪和育肥猪发病率可达100%,成年母猪为15%～19%,以哺乳仔猪受害最严重,死亡率可达95%以上,而成年猪死亡率在10%以下。PEDV主要通过被感染猪只排出的粪便或污染物经口自然感染,新生仔猪潜伏期为24-36小时,肥育猪为2日以上,病猪腹泻开始时排黄色粘稠便,以后变成水样便,腹泻的同时伴有精神沉郁,厌食,消瘦及衰竭。年龄越小,症状越严重,死亡率越高。PED在世界上分布很广,呈现地方性流行性。近年来,流行区域有逐渐扩大的趋势,危害比较严重,给养猪业带来重要经济损失。
本研究首先克隆了PEDV LJB/03株s、n基因,并对其分子特征、遗传变异作出分析,结果显示,不同毒株间的变异率很小,表现出了高度的保守性,适合用作基因工程疫苗研究。针对目前PEDV保护性抗原还不清楚的现状,试验中利用实验室构建的重组大肠杆菌,诱导并纯化了PEDV S蛋白的各个片段s1(22-505aa)、S2(488-980aa)、S34(951-1383aa)、PS420(461-638aa)、S22(502-792aa)以及N蛋白,之后利用纯化蛋白免疫试验兔制备高免血清,以高免血清做中和试验,结果显示,除N蛋白外,所有重组蛋白的抗血清的均具有一定的中和活性,中和效价在1:23～1:93之间,其中以S1、S22、PS420的抗血清中和活性较高,分别可达1:93、1:74、1:89;S34中和效价为1:52;S2抗血清中和效价较低,为1:23;抗血清组合后中和效价呈现不同程度的提高,其中以S1、S22、PS420组合抗血清中和活性最好,可达1:363:S1、S2、S34抗血清组合中和活性为1:199,S22与PS420抗血清组合中和活性1:186。从而筛选出适合于基因工程疫苗研究的基因片段。
为探讨大肠杆菌不耐热肠毒素B单位(LTB)在乳酸菌传递系统的佐剂活性,并进一步提高PS420的免疫原性,试验中克隆了LTB基因并与ps420基因融合,命名为lp0。
以筛选到的免疫保护性抗原蛋白的基因ps1(包含S1、PS420、S22)、ps420、lp0、n为靶基因,分别亚克隆至细胞表面表达型乳酸菌载体pPG-1及分泌表达型乳酸菌载体pPG-2中,电转化后,构建了表达PEDV免疫保护性抗原蛋白的重组干酪乳杆菌表达系统。
将获得的8种重组干酪乳杆菌以2%乳糖为诱导剂进行诱导表达。细胞表面表达型重组干酪乳杆菌诱导后经Western blot、间接免疫荧光、全菌体ELISA试验证实,蛋白获得表达,并且定位于干酪乳杆菌菌体表面;分泌表达型重组干酪乳杆菌经诱导后,Western blot、间接ELISA实验表明,重组的目的蛋白获得了分泌表达。
为探讨表达重组的重组干酪乳杆菌系统作为活菌疫苗潜在的应用价值,本实验以BALB/c小鼠为实验动物,进行免疫学研究。共分为pPG-1-lp0/L.casei393免疫组、pPG-2-lp0/L.casei393免疫组、pPG-1-ps420/L.casei393免疫组、pPG-2-ps420/L.casei393免疫组、pPG-1-n/L.casei393免疫组、pPG-2-n/L.casei393免疫组、pPG-1-ps1/L.casei393免疫组、pPG-2-ps1/L.casei393免疫组、pPG-1联合免疫组(pPG-1-n/L.casei393、pPG-1-ps1/L.casei393)、pPG-2联合免疫组(pPG-2-n/L.casei393、pPG-2-ps1/L.casei393)、pPG-1/L.casei393免疫对照组、pPG-1/L.casei393免疫对照组、PBS对照组,共13组。口服接种10~9活菌量,每2周免疫一次,共免疫三次,每次连续免疫3d,一天免疫一次。免疫后分别于0d、7d、21d、35d、49d、63d、77d收集免疫小鼠新鲜粪便、血液、眼冲洗物、鼻腔冲洗液、外生殖道冲洗液等样品。分别测定样品中特异性的sIgA以及小鼠血清中IgG;用MTT法检测免疫小鼠细胞脾淋巴细胞增殖情况,并检测小鼠脾细胞培养上清中细胞因子IL-4、IFN-γ的水平。
ELISA检测结果表明,口服免疫重组干酪乳杆菌后在小鼠粪便、眼冲洗液、鼻冲洗液、外生殖道冲洗液、肠黏液中均检测到了特异性sIgA,各组一般在免疫后21d即可产生明显的抗体水平(P＜0.05),第35d或49d达到sIgA抗体水平最高峰,与和对照组相比,差异均为显著(P＜0.05)或极显著(P＜0.01),到77d也可检测到抗体存在。同时在小鼠血清中检测到了较高水平的血清抗体,各组一般在免疫后21d即可产生明显的抗体水平(P＜0.05),第35d或49d达到sIgA抗体水平最高峰,和对照组相比,差异均为显著(P＜0.05)或极显著(P＜0.01),到77d也可检测到抗体存在。淋巴细胞增殖试验和细胞因子测定结果显示,免疫组可产生明显的细胞免疫应答。
口服免疫效果表明,该8种重组干酪乳杆菌表达系统均能刺激动物黏膜免疫应答和系统免疫应答,不但在肠道,而且在眼、鼻、外生殖道等部位均可产生分泌型IgA抗体,并可检测到高水平血清IgG;不仅能诱导体液免疫反应,还可诱导细胞免疫效应。中和试验结果表明,血清抗体、肠粘液sIgA均有一定的保护效果,血清抗体中和效价在1:6～1:93之间,肠黏液IgA中和效价1:3～1:35之间。
试验证实细胞表面表达型干酪乳杆菌系统可诱导比分泌表达型干酪乳杆菌系统具有更好的免疫反应;表达ps1基因的重组干酪乳杆菌和表达n基因的重组干酪乳杆菌联合免疫可诱导更强的免疫应答;导向分子LTB的引入增强了重组乳酸菌疫苗的免疫效果,具有佐剂效应。
以上结果说明所构建的重组干酪乳杆菌表达系统作为口服疫苗具有潜在的应用价值,为探索新型PED口服疫苗的研制奠定了基础。
Porcine epidemic diarrhea virus(PEDV),a member of the Coronaviridae,causes an economically important disease in neonatal piglets,which characterized by severe enteritis with anorexia,vomiting,acute diarrhea,dehydration and significant mortality in swine,thereby incurring serious economic losses worldwide due to the death of neonatal piglets and weight loss of the infected pigs.Morbidity and mortality in infected neonatal piglets less than 5 days old approach 100%because of severe diarrhea and dehydration.However,mortality in infected piglets older than 10 days is less than 10%.PED have been reported in many countries,caused serious economic losses due to the death of neonatal piglets and weight loss of the infected pigs.
Lactic acid bacteria(LAB),considered to be safe bacteria with a GRAS(generally regarded as safe) status,possesses many properties that has been thought to be an ideal vaccine which has a good perspective.
The main goals of this thesis were:(1) to understand the knowledge of the data of molecular biology of PEDV strains that are prevalent in China;(2) and select the protective antigen of PEDV; (3) to study the efficacy,immunogenicity of recombinant Lactobacillus casei after oral vaccination.
Above all,we cloned s gene of PEDV LJB/03 strain,then the gene fragment was subcloned into pMD18-T vector,sequenced and analysised.Comparing with PEDV reference strains,S gene had some mutant,and homology of nucleotides and amino acids sequences was between 94.4%and 100%.The LJB/03 n gene has also a high sequence homology to those of other PEDV isolates.The analysis result showed that LJB/03 have high homology with different isolates,so it can serve as target antigen to develop genetic engineering vaccine.
Some of the structural proteins of PEDV carry major epitopes involved in virus neutralization and are essential for the induction of protective humoral responses and the development of an effective vaccine.Rabbit antisera were prepared using full-length N proteins and five truncated expressed fragments covering the S protein fragments[s1(22-505aa),S2(488-980aa), S34(951-1383aa),PS420(461-638aa),S22(502-792aa)].Antisera to S proteins were found to have different neutralizing titres towards PEDV infection in vivo,ranging from 1:23～1:93.Antiserum to the N protein did not contain neutralizing antibodies.Epitopes inducing protective humoral responses to virus infection were located mainly in the region S1,S22,PS420.The in vitro neutralization assay has important implications for the design of an effective vaccine preventing PEDV infection.
The selective gene ps1(including S1,S22,PS420),ps420,n,1p0(LTB and ps420) were subcloned into the expression vector pPG-1 or pPG-2,and then transformed into L.casei 393 by electroporation,resulting in recombinant strain pPG-1-n/L.casei393,pPG-1-ps1/L.casei393,pPG-1 -1p0/L.casei393,pPG-1-ps420/L.casei393,pPG-2-n/L.casei393,pPG-2-ps1/L.casei393,pPG-2-1p0-/L.casei393, pPG-2-ps420/L.casei393,respectively.
The recombinant strains constructed in this study were induced by 2%lactose in MRS medium to express interest protein.The expression protein in the cell-surface expression system (pPG-1-n/L.casei393,pPG-1-ps1/L.casei393,pPG-1-1p0/L.casei393,pPG-1-ps420/L.casei393) was detected by Western blot and the whole bacterica ELISA.The indirect immunofluorescence test showed that the expressed protein was displayed on the cell surface of L.casei 393.
The recombinant strain pPG-2-n/L.casei393,pPG-2-psl/L.casei393,pPG-2-1p0/L.casei393, pPG-2-ps420/L.casei393 was detected via Western blot and indirect ELISA.The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein.The indirect ELISA test also indicated that the interest protein was expressed and secreted into the culture supernatant.
To evaluate the immune responses of recombinant L.casei 393 expressing PEDV protein as oral vaccine,BALB/C mouse were used as animal model immunized with recombinant strains by intragastric administration,and the immune efficacy was analyzed.
The immune protocol was administered on three consecutive days at days 0,1and 2.A booster immunization was given at days 14,15 and 16 and a second booster was given at days 28,29 and 30,the mice were fed with 10~9 recombinant strains.The groups include pPG-1-1p0/L.casei393 immun-ization group,pPG-2-1p0/L.casei393 immunization group,pPG-1-ps420/L.casei393 immunization group,pPG-2-ps420/L.casei393 immunization group,pPG-1-n/L.casei393 imm -unization group,pPG-2-n/L.casei393 immunization group,pPG-1-ps1/L.casei393 immunization group,pPG-2-ps1/L.casei-393 immunization group,pPG1 divalent immunization group(pPG-1-n /L.casei393 and pPG-1-ps1/L.casei393 ),pPG2 divalent immunization group(pPG-2-n/L.casei393 and pPG-2-ps1/L.casei393).As control,the mice were immunized with L.casei393 harboring pPG-2 or pPG-2,or PBS by oral route.
Specific anti-PEDV protein sIgA was detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces,nasal wash,vaginal lavage,eye wash,intestines mucus of mice after intragastric administration,and Specific IgG was detected by indirect ELISA in the serum of immunized mice.The spleen lymphocyte proliferation index(LPI) was measured.IFN-γand IL-4 contents in the supematant of spleen cell culture were also assessed by ELISA.
The results of ELISA showed that the mice immunized with recombinant strains could produce remarkable special slgA level in the feces,nasal wash,vaginal lavage,eye wash of mice on days 21 post oral administration,and highest level were obtained on days 35 or 49 post oral administration. Highest level of anti-PEDV protein IgG in the serum of mice were obtained on days 35 or 49 post oral administration.Special sIgA level in the intestines lavages significant increase.These indicated that the recombinant strains constructed in this study could induce both mucosal immune and system immune responses.The results also demonstrated that recombinant L.casei 393 of cell surface expression type could elicit higher immune response level than that induced by secretion expression type.
The multiple oral immunizations with recombinant L.casei 393 could induce significantly specific mucosal IgA response as well as serum IgG responses.Moreover,there was significant increase of IFN-γand IL-4 contents in the supernatant of spleen cell culture in immunized group. Statistical significant difference was observed among the LPI among these ten immunized groups of mice and control groups.The results demonstrated that recombinant L.casei 393 of secretion expression type could elicit higher immune response level than that induced by cell surface expression type.pPG-1 divalent immunization group(pPG-1-n/L.casei393 and pPG-1-ps1 -/L.casei393),pPG-1 divalent immunization group(pPG-2-n/L.casei393 and pPG-2-ps1 -/L.casei393 ) can induce more effective immune response,leads to synergic effect.
The induced antibodies in serum and intestinal lavages demonstrated neutralizing effect on PEDV infection.Another conclusion is LTB could be an effective advjant in intragastric administeration of recombinant L.casei 393.
Our data has indicated that intragastric administration of the recombinant L.casei 393 could induce specific immune response against PEDV.Our work established a good foundation for further study on the new and effective PEDV recombinant oral vaccines.
引文
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