旋毛虫mif基因与mcd-1基因的DNA免疫保护作用以及泛素共表达对DNA免疫的影响
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摘要
旋毛虫病(Trichinellosis)是由旋毛虫(Trichinella spiralis)引起的一种危害严重的食源性人兽共患寄生虫病,是肉类食品安全性的一个重要指标。在旋毛虫的长期寄生生活中,分泌了大量具有免疫调节功能的蛋白,对宿主免疫系统进行调节,以逃避宿主免疫应答。这些分子除了其免疫调节功能外,还是潜在的疫苗候选分子,将它们应用于疫苗研究中将具有重要意义。泛素(Ubiquitin, Ub)是一个由76个氨基酸残基组成的小蛋白,普遍存在于真核细胞中且序列高度保守。泛素依赖性的蛋白质降解途径是目前已知的重要的、有高度选择性的蛋白降解途径之一。泛素化可对DNA疫苗的免疫应答产生影响。本研究克隆了2个具有免疫调节功能的旋毛虫分泌蛋白基因——旋毛虫巨噬细胞移动抑制因子(Tsmif)和旋毛虫半胱氨酸蛋白酶抑制因子(Tsmcd-1),构建了pVAX1真核表达载体的DNA疫苗,在BALB/c小鼠体内对DNA免疫保护作用进行了研究。结果显示,Tsmif和Tsmcd-1的共表达能够诱导特异性体液免疫应答和细胞免疫应答,并对旋毛虫感染产生部分的免疫保护。此外,与小鼠泛素的共表达增强了细胞免疫应答,并促进了pVAX1-Tsmif-Tsmcd-1免疫产生的抗感染保护能力。
根据GenBank中发表的旋毛虫mif基因(Tsmif)序列(AY050661)和旋毛虫mcd-1基因(Tsmcd-1)序列(DQ777102),设计引物。以旋毛虫成虫总RNA为模板,RT-PCR扩增出大小2个TsMIF基因片段Tsmif和Tsmif",1个TsMCD-1基因片段Tsmcd-1.构建了3个重组质粒pVAX1-Tsmif、pVAX1-Tsmcd-1和pVAX1-Tsmif-Tsmcd-1;双酶切鉴定结果显示,阳性质粒分别切出3000bp的pVAX1.0质粒片段和大小相符的靶基因片段,即：345bp(Tsmif)、1221bp (Tsmcd-1)和1602bp (Tsmif-Tsmcd-1)。序列分析结果显示,扩增的Tsmif和Tsmif"基因片段与GenBank中已公布序列(AY050661)的核苷酸同源性和氨基酸同源性均为99.7%；扩增的Tsmcd-1基因片段与GenBank中已公布序列(DQ777102)的核苷酸同源性为99%,氨基酸同源性为100%。
以pET28(b)为载体,构建原核表达重组质粒pET28(b)-Tsmif、pET28(b)-Tsmcd-1和pET28(b)-Tsmif-Tsmcd-1,分别在BL21大肠杆菌中表达。SDS-PAGE表明,重组蛋白rTsMIF表达于上清中,而重组蛋白rTsMCD-1与rTsMIF-TsMCD-1则主要以包涵体的形式存在。Western blot分析结果显示3种重组蛋白均能与感染旋毛虫的小鼠血清反应出现特异性条带(16.5kDa、50kDa和63kDa)。重组蛋白经纯化后,分别与弗氏佐剂乳化皮下注射免疫BALB/c小鼠,制备了以上3种重组蛋白的小鼠抗血清。为了检测TsMIF与TsMCD-1的特异性抗体,以重组蛋白rTsMIF-TsMCD-1作为抗原建立了间接ELISA(rTsMIF-TsMCD-1-E LISA);经方阵试验确定抗原包被浓度为2.3μml,血清检测稀释度为1：128,抗原与抗体的最佳反应时间为90min。
为了研究Tsmif和Tssmcd-1的DNA疫苗免疫效果,将三种重组质粒pVAX1-Tsmif, pVAX1-Tsmcd-1和pVAX1-Tsmif-Tsmcd-1分别用肌肉注射的方式免疫BALB/c小鼠,以PBS和pVAX1分别为空白对照和空载体质粒对照；每组20只BALB/c小鼠,共免疫2次,间隔2周。二免后14d,进行攻虫保护试验。经口感染旋毛虫感染性幼虫(L1)35d后,剖杀所有小鼠分别计算肌肉荷虫量(LPG)。目的基因在小鼠体内的表达分别用RT-PCR和Western blot进行鉴定。结果显示,目的基因均在肌肉注射部位获得了表达,pVAX1-Tsmif-Tsmcd-1免疫产生了TsMIF和TsMCD-1的共表达蛋白。在首免后0、10、20、28d采集免疫小鼠血清,用重组蛋白rTsMIF-TsMCD-1包被的间接ELISA对免疫小鼠的特异性抗体水平的动态变化进行了分析,结果显示,pVAX1-Tsmif免疫没有产生特异性抗体应答；而pVAX1-Tsmcd-1和pVAX1-Tsmif-Tsmcd-1免疫分别诱导了特异性IgG.IgM和IgA的产生,但没有检测到特异性IgE抗体。与pVAX1-Tsmcd-1相比,pVAX1-Tsmif-Tsmcd-1免疫引起了较低的抗体应答(P<0.05)。IgG亚型分析显示,IgG2a和IgG2b的抗体滴度显著高于IgG1,表现为Thl型抗体占优势的体液免疫应答。此外,对血清细胞因子含量的动态变化的分析结果显示,三种重组质粒的DNA免疫均产生了一定水平的Thl型细胞因子(IFN-γ),但未检测到Th2型细胞因子(IL-4、IL-5);低水平的TGF-β1可能与抑制优势细胞因子(IFN-γ)有关；各组均未能检测到IL-17,暗示没有炎症反应的发生。二免后14d,用流式细胞分析仪分析了免疫小鼠外周血淋巴细胞中CD4+T细胞和CD8+T细胞的数量,结果显示,3个重组质粒免疫组的CD4+T细胞数量显著高于对照组(P<0.05),特别是pVAX1-Tsmif-Tsmcd-1免疫组(P<0.01);而pVAX1-Tsmif和pVAX1-Tsmif-Tsmcd-1免疫组的CD8+T细胞的数量也显著高于对照组(P<0.05),pVAX1-Tsmcd-1免疫组则与对照无差异(P>0.05)；3个重组质粒免疫的CD4+/CD8+值均高于对照组,说明重组质粒免疫均促进了小鼠T淋巴细胞的增殖。攻虫保护试验结果显示,单独的pVAX1-Tsmif和pVAX1-Tsmcd-1免疫均未能获得LPG的减少,而pVAX1-Tsmif-Tsmcd-1免疫组的LPG则显著低于对照组(P<0.05),获得了23.17%的肌幼虫减虫率,表现出部分的免疫保护性。
为了研究靶抗原泛素化对DNA免疫的影响,我们用RT-PCR克隆了小鼠泛素基因(Ub),并构建了与Ub共表达的重组质粒pVAX1-Ub-Tsmif、pVAX1-Ub-Tsmcd-1和pVAX1-Ub-Tsmif-Tsmcd-1。将以上重组质粒分别转染BHK细胞,Western blot没有检测到与重组蛋白TsMIF-TsMCD-1抗血清反应的目的条带,但加入蛋白酶体抑制因子MG-132后,分别检测到了大约22kDa、57kDa和68kDa的条带,其大小与预测的泛素化蛋白Ub-TsMIF、Ub-TsMCD-1和Ub-TsMIF-TsMCD-1的大小相符。体外转染试验结果显示,Ub共表达重组质粒转染BHK细胞后,表达了靶蛋白(TsMIF、TsMCD-1或TsMIF-TsMCD-1)的泛素化蛋白；泛素化促进了靶蛋白在BHK细胞内的降解,而MG-132可以抑制这种降解作用。随后,将Ub共表达重组质粒分别用肌肉注射的方式免疫BALB/c小鼠,以PBS和pVAX1、pVAX1-Ub分别为空白对照和空载体质粒对照、泛素免疫对照。通过检测特异性抗体应答、血清细胞因子水平和外周血CD4+T细胞和CD8+T细胞的数量,以及攻虫保护作用,对Ub共表达重组质粒的DNA免疫效果进行了分析。结果显示,靶抗原的泛素化下调了体液免疫应答,却促进了Thl型细胞因子的分泌,以及CD8+T细胞的增殖和免疫小鼠脾细胞的特异性CTL活性；重要的是,pVAX1-Ub-Tsmif-Tsmcd-1免疫组获得了更强的免疫保护性,表现为38%的肌幼虫减虫率。同时,这些结果也反映了Thl型细胞因子和CD8+T细胞免疫应答对旋毛虫抗感染的积极促进作用。
Trichinella spiralis, the etiologic agent of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasitic diseases worldwide. DNA vaccination has become an increasingly attractive approach for vaccination against T. spiralis infection. The parasite can persist in muscle tissues for many years through its ability to produce a variety of production of biologically active proteins to suppress the host immune response. The immunomodulatory functions of excretory-secretory (ES) products during T. spiralis infection and their roles in establishing parasitism in the host have led researchers to explore their potential as a vaccine target. Ubiquitin is a highly conserved76-amino-acid polypeptide in the cytoplasm of eukaryotic cells. Ubiquitin/proteasome-dependent pathway is a highly selective proteolysis pathway. Ubiquitination has been used in DNA vaccination. In this study, plasmids expressing/co-expressing macrophage migration inhibitory factor (MIF), multi cystatin-like domain protein (MCD-1) of T. spiralis (TsMIF and TsMCD-1) were constructed. Their ability to generate a protective immune response against T. spiralis infection was evaluated in BALB/c mice. Results of DNA vaccination showed Thl immune response, and the pVAXl-Tsmif-Tsmcd-1vaccine induce a partial protection against infection. In addition, coexpression with Ub enhanced the Thl cytokines production and CD8+T cells immune response, as well as the protection against T. spiralis infection induced by pVAXl-Tsmif-Tsmcd-1vaccine.
According to GenBank sequences of Tsmif(AY050661) and Tsmcd-1(DQ777102), five primers were designed and used to clone two gene segments of MIF(Tsmif and Tsmif") and one gene segment of MCD-1(Tsmcd-1) with RT-PCR from adult worm of Trichinella spiralis. Three recombinant plasmids of pVAX1-Tsmif, pVAXl-Tsmcd-1and pVAX1-Tsmif-Tsmcd-1were constructed respectively. Digestion identification showed two segments of3000bp and345bp,3000bp and1221bp,3000bp and1602bp, respectively. Comparisons of sequences of Tsmif and Tsmif " with the GenBank sequence of Tsmif (AY050661) indicated nucleotide and amino acid sequence homologies of99.7%respectively. Comparisons of the sequence of Tsmcd-1with the GenBank sequence of Tsmcd-I (DQ777102) indicated nucleotide and amino acid sequence homologies of99%and100%.
Three recombinant plasmids of pET28(b)-Tsmif, pET28(b)-Tsmcd-1and pET28(b)-Tsmif-Tsmcd-1were constructed and expressed in Escherichia. coli BL21. SDS-PAGE showed that the recombinant protein of rTsMIF existed in supernant as a soluble protein; recombinant proteins of rTsMCD-1and rTsMIF-TsMCD-1mainly existed in inclusion bodies. Western blot showed bands of16.5kDa,50kDa and63kDa respectively, suggesting that these three recombinant proteins could react with serum of mice infected with Trichinella spiralis. To produce anti-serum, purified recombinant proteins were mixed with FCA (freund's complete adjuvant) and subcutaneously injected BALB/c mice respectively. A rTsMIF-TsMCD-1-based indirect enzyme-linked immunosorbent assay (rTsMIF-TsMCD-1-ELISA) was developed for detecting specific antibodies against TsMIF and TsMCD-1. The conditions of rTsMIF-TsMCD-1-ELISA were determined as follows:2.3μg/ml of rTsMIF-TsMCD-1used to coat ELISA plate,1:128of sera as detecting samples and serum sample being incubated at37℃for90minutes.
DNA vaccination of three recombinant plasmids expressing/co-expressing TsMIF and TsMCD-1were performed by intramscular injection in BALB/c mice respectively, including pVAX1-Tsmif, pVAX1-Tsmcd-1, or pVAX1-Tsmif-Tsmcd-1, with controls of PBS and pVAXl. Groups of20mice were immunized twice at two-week intervals. Fourteen days past the second vaccination, challenge was carried out by oral infection with200L1infectious T. sipralis larvae per animal. Thirty-five days later,10mice in every group were killed to detect LPG (larvae per gram) and reduction ratio of muscle larvae. Detection of in vivo target gene expression was performed by RT-PCR and Western blot, indicating that target genes were expressed in injected muscle but not in contralateral noninjected muscle, and coexpression protein of TsMIF and TsMCD-1was found in mice vaccinated with pVAX1-Tsmif-Tsmcd-1. Sera collected0,10,20and28days after the first vaccination were assayed for the presence of specific antibodies against recombinant TsMIF-TsMCD-1protein. Results showed that vaccination with pVAXl-Tsmif failed to induce specific antibodies and pVAX1-Tsmcd-1or pVAXl-Tsmif-Tsmcd-1vaccine induced specific antibodies of IgG, IgM and IgA, but no IgE. Levels of specific antibodies in mice vaccinated with pVAX1-Tsmif-Tsmcd-1were less than pVAX1-Tsmcd-1(P<0.05). Both IgG2a and IgG2b showed higher proportion than IgG1(P<0.05) in these two groups, showing a Thl humoral immune response. Concentrations of five serum cytokines were detected and showed higher levels of Thl cytokines (IFN-y), no Th2cytokines (IL-4, IL-5). The lower levels of TGF-β1might be related to suppressing predominant cytokines (IFN-y). The failure to detection of IL-17displayed the absence of inflammation. Fourteen days past the second vaccination, the percentages of CD4+T cells (CD4+%) and CD8+T cells (CD8+%) in peripheral blood lymphocytes were determined, showing more CD4+%in all of the three recombinant plasmids groups than PBS control (P<0.05), especially pVAX1-Tsmif-Tsmcd-1vaccine (P<0.01). The percentage of CD8+T cells in mice vaccinated with pVAX1-Tsmif and pVAX1-Tsmif-Tsmcd-1respectively was more than PBS control (P<0.05), but no significant difference was observed in pVAXl-Tsmif vaccination. Ratio of CD4+%to CD8+%in either of the three recombinant plasmids groups was higher than PBS control (P<0.05), indicating the enhancement of proliferation of mouse T cells. Challenge infection demonstrated that no reduction of LPG was observed in mice vaccinated with pVAX1-Tsmif or pVAX1-Tsmcd-1, but immunization with pVAX1-Tsmif-Tsmcd-1reduced worm burdens by23.17%versus controls (P<0.05) suggesting a partial immune protection against T. spiralis.
To confirm the influence of ubiquitination on DNA vaccination, the Ub gene of mouse was cloned with RT-PCR and three recombinant plasmids of pVAX1-Ub-Tsmif, pVAX1-Ub-Tsmcd-1and pVAX1-Ub-Tsmif-Tsmcd-1were constructed respectively. These recombinant Ub-coexpression plasmids were transformed in BHK cells, and Western blot analysis failed to detect target bands respond to anti-serum of recombinant protein TsMIF-TsMCD-1. However, after adding the proteasome inhibitor MG-132into transformed cells, bands of22kDa,57kDa and68kDa were observed respecitvly, which were consistent with the sizes of coexpression proteins of Ub-TsMIF, Ub-TsMCD-1and Ub-TsMIF-TsMCD-1, respecitvly. These results showed that proteins coexpressing mouse ubiquitin (8.57kDa) and target protein (TsMIF, TsMCD-1, and TsMIF-TsMCD-1) were expressed in BHK cells transformed with the Ub-coexpression plasmids; ubiquitination promoted the degradation of target proteins in BHK cells and MG-132could inhibit the degradation. Later, DNA vaccination with these recombinant Ub-coexpression plasmids was performed in BALB/c mice, with the controls of PBS, pVAXl and pVAX1-Ub. After detection of specific antibodies response, serum cytokines and CD4+or CD8+T cells, as well as the protection against T. spiralis challenge infection, the immunity efficacies of these Ub-coexpression plasmids were evaluated. The results displayed that Ubiquitination reduced humarol immune responses, but promoted Thl cytokines production, CD8+T cells prolifecation and the specific CTL response in vaccinated mice. Importantly, a stronger reduction of LPG was induced in mice vaccinated with pVAXl-Ub-Tsmif-Tsmcd-1. These results also suggested that the stronger Thl cytokines and CD8+T cells immune response induced by ubiquination might play an active role in immune protection against T. spiralis.
根据GenBank中发表的旋毛虫mif基因(Tsmif)序列(AY050661)和旋毛虫mcd-1基因(Tsmcd-1)序列(DQ777102),设计引物。以旋毛虫成虫总RNA为模板,RT-PCR扩增出大小2个TsMIF基因片段Tsmif和Tsmif",1个TsMCD-1基因片段Tsmcd-1.构建了3个重组质粒pVAX1-Tsmif、pVAX1-Tsmcd-1和pVAX1-Tsmif-Tsmcd-1;双酶切鉴定结果显示,阳性质粒分别切出3000bp的pVAX1.0质粒片段和大小相符的靶基因片段,即：345bp(Tsmif)、1221bp (Tsmcd-1)和1602bp (Tsmif-Tsmcd-1)。序列分析结果显示,扩增的Tsmif和Tsmif"基因片段与GenBank中已公布序列(AY050661)的核苷酸同源性和氨基酸同源性均为99.7%；扩增的Tsmcd-1基因片段与GenBank中已公布序列(DQ777102)的核苷酸同源性为99%,氨基酸同源性为100%。
以pET28(b)为载体,构建原核表达重组质粒pET28(b)-Tsmif、pET28(b)-Tsmcd-1和pET28(b)-Tsmif-Tsmcd-1,分别在BL21大肠杆菌中表达。SDS-PAGE表明,重组蛋白rTsMIF表达于上清中,而重组蛋白rTsMCD-1与rTsMIF-TsMCD-1则主要以包涵体的形式存在。Western blot分析结果显示3种重组蛋白均能与感染旋毛虫的小鼠血清反应出现特异性条带(16.5kDa、50kDa和63kDa)。重组蛋白经纯化后,分别与弗氏佐剂乳化皮下注射免疫BALB/c小鼠,制备了以上3种重组蛋白的小鼠抗血清。为了检测TsMIF与TsMCD-1的特异性抗体,以重组蛋白rTsMIF-TsMCD-1作为抗原建立了间接ELISA(rTsMIF-TsMCD-1-E LISA);经方阵试验确定抗原包被浓度为2.3μml,血清检测稀释度为1：128,抗原与抗体的最佳反应时间为90min。
为了研究Tsmif和Tssmcd-1的DNA疫苗免疫效果,将三种重组质粒pVAX1-Tsmif, pVAX1-Tsmcd-1和pVAX1-Tsmif-Tsmcd-1分别用肌肉注射的方式免疫BALB/c小鼠,以PBS和pVAX1分别为空白对照和空载体质粒对照；每组20只BALB/c小鼠,共免疫2次,间隔2周。二免后14d,进行攻虫保护试验。经口感染旋毛虫感染性幼虫(L1)35d后,剖杀所有小鼠分别计算肌肉荷虫量(LPG)。目的基因在小鼠体内的表达分别用RT-PCR和Western blot进行鉴定。结果显示,目的基因均在肌肉注射部位获得了表达,pVAX1-Tsmif-Tsmcd-1免疫产生了TsMIF和TsMCD-1的共表达蛋白。在首免后0、10、20、28d采集免疫小鼠血清,用重组蛋白rTsMIF-TsMCD-1包被的间接ELISA对免疫小鼠的特异性抗体水平的动态变化进行了分析,结果显示,pVAX1-Tsmif免疫没有产生特异性抗体应答；而pVAX1-Tsmcd-1和pVAX1-Tsmif-Tsmcd-1免疫分别诱导了特异性IgG.IgM和IgA的产生,但没有检测到特异性IgE抗体。与pVAX1-Tsmcd-1相比,pVAX1-Tsmif-Tsmcd-1免疫引起了较低的抗体应答(P<0.05)。IgG亚型分析显示,IgG2a和IgG2b的抗体滴度显著高于IgG1,表现为Thl型抗体占优势的体液免疫应答。此外,对血清细胞因子含量的动态变化的分析结果显示,三种重组质粒的DNA免疫均产生了一定水平的Thl型细胞因子(IFN-γ),但未检测到Th2型细胞因子(IL-4、IL-5);低水平的TGF-β1可能与抑制优势细胞因子(IFN-γ)有关；各组均未能检测到IL-17,暗示没有炎症反应的发生。二免后14d,用流式细胞分析仪分析了免疫小鼠外周血淋巴细胞中CD4+T细胞和CD8+T细胞的数量,结果显示,3个重组质粒免疫组的CD4+T细胞数量显著高于对照组(P<0.05),特别是pVAX1-Tsmif-Tsmcd-1免疫组(P<0.01);而pVAX1-Tsmif和pVAX1-Tsmif-Tsmcd-1免疫组的CD8+T细胞的数量也显著高于对照组(P<0.05),pVAX1-Tsmcd-1免疫组则与对照无差异(P>0.05)；3个重组质粒免疫的CD4+/CD8+值均高于对照组,说明重组质粒免疫均促进了小鼠T淋巴细胞的增殖。攻虫保护试验结果显示,单独的pVAX1-Tsmif和pVAX1-Tsmcd-1免疫均未能获得LPG的减少,而pVAX1-Tsmif-Tsmcd-1免疫组的LPG则显著低于对照组(P<0.05),获得了23.17%的肌幼虫减虫率,表现出部分的免疫保护性。
为了研究靶抗原泛素化对DNA免疫的影响,我们用RT-PCR克隆了小鼠泛素基因(Ub),并构建了与Ub共表达的重组质粒pVAX1-Ub-Tsmif、pVAX1-Ub-Tsmcd-1和pVAX1-Ub-Tsmif-Tsmcd-1。将以上重组质粒分别转染BHK细胞,Western blot没有检测到与重组蛋白TsMIF-TsMCD-1抗血清反应的目的条带,但加入蛋白酶体抑制因子MG-132后,分别检测到了大约22kDa、57kDa和68kDa的条带,其大小与预测的泛素化蛋白Ub-TsMIF、Ub-TsMCD-1和Ub-TsMIF-TsMCD-1的大小相符。体外转染试验结果显示,Ub共表达重组质粒转染BHK细胞后,表达了靶蛋白(TsMIF、TsMCD-1或TsMIF-TsMCD-1)的泛素化蛋白；泛素化促进了靶蛋白在BHK细胞内的降解,而MG-132可以抑制这种降解作用。随后,将Ub共表达重组质粒分别用肌肉注射的方式免疫BALB/c小鼠,以PBS和pVAX1、pVAX1-Ub分别为空白对照和空载体质粒对照、泛素免疫对照。通过检测特异性抗体应答、血清细胞因子水平和外周血CD4+T细胞和CD8+T细胞的数量,以及攻虫保护作用,对Ub共表达重组质粒的DNA免疫效果进行了分析。结果显示,靶抗原的泛素化下调了体液免疫应答,却促进了Thl型细胞因子的分泌,以及CD8+T细胞的增殖和免疫小鼠脾细胞的特异性CTL活性；重要的是,pVAX1-Ub-Tsmif-Tsmcd-1免疫组获得了更强的免疫保护性,表现为38%的肌幼虫减虫率。同时,这些结果也反映了Thl型细胞因子和CD8+T细胞免疫应答对旋毛虫抗感染的积极促进作用。
Trichinella spiralis, the etiologic agent of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasitic diseases worldwide. DNA vaccination has become an increasingly attractive approach for vaccination against T. spiralis infection. The parasite can persist in muscle tissues for many years through its ability to produce a variety of production of biologically active proteins to suppress the host immune response. The immunomodulatory functions of excretory-secretory (ES) products during T. spiralis infection and their roles in establishing parasitism in the host have led researchers to explore their potential as a vaccine target. Ubiquitin is a highly conserved76-amino-acid polypeptide in the cytoplasm of eukaryotic cells. Ubiquitin/proteasome-dependent pathway is a highly selective proteolysis pathway. Ubiquitination has been used in DNA vaccination. In this study, plasmids expressing/co-expressing macrophage migration inhibitory factor (MIF), multi cystatin-like domain protein (MCD-1) of T. spiralis (TsMIF and TsMCD-1) were constructed. Their ability to generate a protective immune response against T. spiralis infection was evaluated in BALB/c mice. Results of DNA vaccination showed Thl immune response, and the pVAXl-Tsmif-Tsmcd-1vaccine induce a partial protection against infection. In addition, coexpression with Ub enhanced the Thl cytokines production and CD8+T cells immune response, as well as the protection against T. spiralis infection induced by pVAXl-Tsmif-Tsmcd-1vaccine.
According to GenBank sequences of Tsmif(AY050661) and Tsmcd-1(DQ777102), five primers were designed and used to clone two gene segments of MIF(Tsmif and Tsmif") and one gene segment of MCD-1(Tsmcd-1) with RT-PCR from adult worm of Trichinella spiralis. Three recombinant plasmids of pVAX1-Tsmif, pVAXl-Tsmcd-1and pVAX1-Tsmif-Tsmcd-1were constructed respectively. Digestion identification showed two segments of3000bp and345bp,3000bp and1221bp,3000bp and1602bp, respectively. Comparisons of sequences of Tsmif and Tsmif " with the GenBank sequence of Tsmif (AY050661) indicated nucleotide and amino acid sequence homologies of99.7%respectively. Comparisons of the sequence of Tsmcd-1with the GenBank sequence of Tsmcd-I (DQ777102) indicated nucleotide and amino acid sequence homologies of99%and100%.
Three recombinant plasmids of pET28(b)-Tsmif, pET28(b)-Tsmcd-1and pET28(b)-Tsmif-Tsmcd-1were constructed and expressed in Escherichia. coli BL21. SDS-PAGE showed that the recombinant protein of rTsMIF existed in supernant as a soluble protein; recombinant proteins of rTsMCD-1and rTsMIF-TsMCD-1mainly existed in inclusion bodies. Western blot showed bands of16.5kDa,50kDa and63kDa respectively, suggesting that these three recombinant proteins could react with serum of mice infected with Trichinella spiralis. To produce anti-serum, purified recombinant proteins were mixed with FCA (freund's complete adjuvant) and subcutaneously injected BALB/c mice respectively. A rTsMIF-TsMCD-1-based indirect enzyme-linked immunosorbent assay (rTsMIF-TsMCD-1-ELISA) was developed for detecting specific antibodies against TsMIF and TsMCD-1. The conditions of rTsMIF-TsMCD-1-ELISA were determined as follows:2.3μg/ml of rTsMIF-TsMCD-1used to coat ELISA plate,1:128of sera as detecting samples and serum sample being incubated at37℃for90minutes.
DNA vaccination of three recombinant plasmids expressing/co-expressing TsMIF and TsMCD-1were performed by intramscular injection in BALB/c mice respectively, including pVAX1-Tsmif, pVAX1-Tsmcd-1, or pVAX1-Tsmif-Tsmcd-1, with controls of PBS and pVAXl. Groups of20mice were immunized twice at two-week intervals. Fourteen days past the second vaccination, challenge was carried out by oral infection with200L1infectious T. sipralis larvae per animal. Thirty-five days later,10mice in every group were killed to detect LPG (larvae per gram) and reduction ratio of muscle larvae. Detection of in vivo target gene expression was performed by RT-PCR and Western blot, indicating that target genes were expressed in injected muscle but not in contralateral noninjected muscle, and coexpression protein of TsMIF and TsMCD-1was found in mice vaccinated with pVAX1-Tsmif-Tsmcd-1. Sera collected0,10,20and28days after the first vaccination were assayed for the presence of specific antibodies against recombinant TsMIF-TsMCD-1protein. Results showed that vaccination with pVAXl-Tsmif failed to induce specific antibodies and pVAX1-Tsmcd-1or pVAXl-Tsmif-Tsmcd-1vaccine induced specific antibodies of IgG, IgM and IgA, but no IgE. Levels of specific antibodies in mice vaccinated with pVAX1-Tsmif-Tsmcd-1were less than pVAX1-Tsmcd-1(P<0.05). Both IgG2a and IgG2b showed higher proportion than IgG1(P<0.05) in these two groups, showing a Thl humoral immune response. Concentrations of five serum cytokines were detected and showed higher levels of Thl cytokines (IFN-y), no Th2cytokines (IL-4, IL-5). The lower levels of TGF-β1might be related to suppressing predominant cytokines (IFN-y). The failure to detection of IL-17displayed the absence of inflammation. Fourteen days past the second vaccination, the percentages of CD4+T cells (CD4+%) and CD8+T cells (CD8+%) in peripheral blood lymphocytes were determined, showing more CD4+%in all of the three recombinant plasmids groups than PBS control (P<0.05), especially pVAX1-Tsmif-Tsmcd-1vaccine (P<0.01). The percentage of CD8+T cells in mice vaccinated with pVAX1-Tsmif and pVAX1-Tsmif-Tsmcd-1respectively was more than PBS control (P<0.05), but no significant difference was observed in pVAXl-Tsmif vaccination. Ratio of CD4+%to CD8+%in either of the three recombinant plasmids groups was higher than PBS control (P<0.05), indicating the enhancement of proliferation of mouse T cells. Challenge infection demonstrated that no reduction of LPG was observed in mice vaccinated with pVAX1-Tsmif or pVAX1-Tsmcd-1, but immunization with pVAX1-Tsmif-Tsmcd-1reduced worm burdens by23.17%versus controls (P<0.05) suggesting a partial immune protection against T. spiralis.
To confirm the influence of ubiquitination on DNA vaccination, the Ub gene of mouse was cloned with RT-PCR and three recombinant plasmids of pVAX1-Ub-Tsmif, pVAX1-Ub-Tsmcd-1and pVAX1-Ub-Tsmif-Tsmcd-1were constructed respectively. These recombinant Ub-coexpression plasmids were transformed in BHK cells, and Western blot analysis failed to detect target bands respond to anti-serum of recombinant protein TsMIF-TsMCD-1. However, after adding the proteasome inhibitor MG-132into transformed cells, bands of22kDa,57kDa and68kDa were observed respecitvly, which were consistent with the sizes of coexpression proteins of Ub-TsMIF, Ub-TsMCD-1and Ub-TsMIF-TsMCD-1, respecitvly. These results showed that proteins coexpressing mouse ubiquitin (8.57kDa) and target protein (TsMIF, TsMCD-1, and TsMIF-TsMCD-1) were expressed in BHK cells transformed with the Ub-coexpression plasmids; ubiquitination promoted the degradation of target proteins in BHK cells and MG-132could inhibit the degradation. Later, DNA vaccination with these recombinant Ub-coexpression plasmids was performed in BALB/c mice, with the controls of PBS, pVAXl and pVAX1-Ub. After detection of specific antibodies response, serum cytokines and CD4+or CD8+T cells, as well as the protection against T. spiralis challenge infection, the immunity efficacies of these Ub-coexpression plasmids were evaluated. The results displayed that Ubiquitination reduced humarol immune responses, but promoted Thl cytokines production, CD8+T cells prolifecation and the specific CTL response in vaccinated mice. Importantly, a stronger reduction of LPG was induced in mice vaccinated with pVAXl-Ub-Tsmif-Tsmcd-1. These results also suggested that the stronger Thl cytokines and CD8+T cells immune response induced by ubiquination might play an active role in immune protection against T. spiralis.
引文
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