弓形虫新基因表位疫苗质粒的构建及鉴定

目的构建两个弓形虫新基因2B9G1、7C3-C3的表位疫苗质粒,为阐明表位疫苗的保护性奠定基础。方法采用生物信息学方法对新基因2B9G1、7C3-C3进行表位预测,PCR扩增基因中编码3个表位的片段W2b、W2a和W4a,连接入pcDNA3,转入DH5α,挑阳性菌落进行PCR、酶切和测序鉴定;同法将W2a、W4a分别克隆入pcDNA3-W2b载体中W2b片段下游,再将W4a克隆入pcDNA3-W2b2a载体中W2a片段下游。结果经PCR、酶切鉴定及序列测定,W2b、W2a和W4a 3个片段分别插入pcDNA3预期位置;W2a和W4a片段分别插入pcDNA3-W2b预期位置;W4a片段插入pcDNA3-W2b2a预期位置。结论成功构建单表位、双表位、多表位疫苗质粒pcDNA3-W2b、pcDNA3-W2a、pcDNA3-W4a、pcDNA3-W2b2a、pcDNA3-W2b4a、pcDNA3-W2a2b4a。     

弓形虫影响前列腺素E2合成的细胞内信号调节途径

目的　探讨弓形虫感染巨噬细胞过程中花生四烯酸 (AA)及前列腺素E₂ (PGE₂ )的产生及其相关的细胞内信号调节途径。　方法　构建刚地弓形虫 小鼠巨噬细胞侵染模型 ,应用气相色谱、酶联免疫吸附测定 (ELISA)、逆转录 聚合酶链反应 (RT-PCR)和蛋白质印迹法 (Westernblotting)检测钙离子调节剂乙二醇双 ( 2-氨基乙醚 )四乙酸 (EG-TA)、钙离子螯合剂 (BAPTA/AM )、钙调蛋白抑制剂三氟拉嗪 (TFP)及蛋白激酶C (PKC)抑制剂 1-( 5-磺酰基 ) 异喹啉-2-甲基-哌嗪盐酸盐 (H7)对弓形虫诱导的AA、PGE₂ 含量及促细胞分裂剂诱导性环加氧酶-2 (COX-2 )mRNA和蛋白质表达水平的影响。　结果　EGTA、BAPTA/AM及TFP均可抑制弓形虫诱导的巨噬细胞AA和PGE₂ 合成 ;PKC抑制剂H7可明显抑制弓形虫和脂多糖 (LPS)诱导的巨噬细胞PGE₂ 合成 ,并伴随着COX-2mRNA和蛋白质表达呈剂量依赖性减少。　结论　弓形虫侵染巨噬细胞过程可能通过钙信号转导途径调节COX-2底物AA的含量和PKC依赖的COX-2代谢途径调节PGE₂ 的合成。     

孕中期弓形虫感染对孕鼠血清孕酮及雌三醇含量的影响

目的探讨孕中期弓形虫感染对孕鼠血清孕酮(progesterone)及雌三醇(estriol)含量的影响。方法孕中期BALB/c孕鼠随机分为染虫组和健康对照组,染虫组经腹腔无菌接种纯化的RH强毒株弓形虫速殖子,健康对照组注射等量0.01mol/LPBS。分别于妊娠第10、12、14、16、18d,摘孕鼠眼球取血,经放射免疫法测定血清孕酮和雌三醇含量。结果在孕第10、12、14d,孕鼠血清孕酮和雌三醇含量染虫组与对照组比较差异无统计学意义(P>0.05);在孕第16、18d,两组比较差异有统计学意义(P<0.01)。结论孕中期弓形虫感染可引起孕鼠血清孕酮和雌三醇含量下降,这可能是弓形虫感染致胚胎发育迟缓、胎盘组织炎性病变及滋养层细胞凋亡增加的结果,也可能是胚胎发育迟缓、滋养层细胞凋亡增加的原因。     

弓形虫致密颗粒蛋白GRA2真核表达质粒的构建与体外表达

目的构建弓形虫致密颗粒蛋白GRA2的真核表达重组质粒。方法设计合成GRA2引物，运用PCR方法扩增其基因片段，经克隆至pMDl8-T载体后，亚克隆至真核表达质粒pcDNA3．1（-）而构建重组表达质粒pcDNA3．1-GRA2。脂质体法将构建的重组质粒转染HFF细胞，RT—PCR法检测转染细胞中GRA2的表达情况。结果PCR扩增GRA2基因序列正确，构建的重组表达质粒pcDNA3．1-GRA2经PCR、EcoRⅠ／HindⅢ双酶切和测序鉴定正确；转染GRA2基因的细胞，RT—PCR可见目的条带。结论成功获得真核表达重组质粒pcDNA3．1-GRA2，为进一步研究弓形虫疫苗的免疫保护性奠定基础。     

Different effect of Toxoplasma gondii infection on adhesion capacity of fibroblasts and monocytes

This study investigated the effect of infection with the apicomplexan parasite Toxoplasma gondii , in combination with the concomitant cytokine environment (IFN-γ/TNF-α), on adhesion of THP-1 monocytic cells to MRC-5 fibroblasts. Surprisingly, infection of THP-1 cells decreased their adhesion to the MRC-5 cell monolayer. This decrease was compensated by IFN-γ/TNF-α stimulation. In contrast, infection of MRC-5 cells significantly increased adhesion, which was synergistically augmented by cytokine stimulation. Levels of ICAM-1 (CD54) on MRC-5 cells, as well as LFA-1 (CD11a) on THP-1 cells, were not changed by infection, neither in resting, nor in cytokine stimulated cells. These results show that T. gondii infection alters adhesion properties and reactivity to cytokine stimulation in a cell-specific way.     

Chinese 1基因型弓形虫棒状体蛋白 ROP16的基因克隆表达及生物信息学分析

目的：构建弓形虫Chinese 1基因型TgCtWh3（以后均简称Wh3）株棒状体蛋白（ rhoptry protein ,ROPs）16的原核和真核重组表达质粒及其3D结构。方法参照ROP16序列分别设计引物,采用PCR从弓形虫Chinese 1基因型Wh3株基因组DNA中扩增出编码ROP16的基因片段,克隆至pMD18-T载体;经PCR及测序分析鉴定;阳性克隆的质粒分别亚克隆至原核表达载体pET-28a和真核表达载体pEGFP-C2,分别转化大肠杆菌BL21和DH5α,PCR和酶切鉴定转化菌落插入的序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE和免疫印迹分析融合蛋白的表达;将构建的真核重组质粒经脂质体转染293T细胞,观察其在细胞中表达;采用生物信息学方法分析构建了蛋白的3 D结构。结果各组均PCR扩增出约2．1 kb ROP16基因的特异片段,序列检测结果均正确;分别亚克隆到原核表达载体pET-28a和真核表达载体pEGFP-C2中,成功构建了Chinese 1基因型弓形虫棒状体蛋白ROP16的原核表达质粒和真核表达质粒;原核表达质粒在大肠杆菌中表达了ROP16的融合蛋白;真核表达质粒在293T细胞中成功表达,成功构建出ROP16基因的3D结构图。结论以pET-28a和pEGFP-C2为载体,分别成功构建并表达了ROP16的原核和真核重组质粒,并构建出其3D结构图。     

Azathioprine-induced toxoplasma gondii infection in a patient with Crohn's disease with NUDT15 variation: A case report

Introduction:Azathioprine (AZA) has been widely used for the treatment of various immune-related diseases and has become a mainstay in the treatment of inflammatory bowel disease. However, patients with genetic mutations may experience severe adverse events when treated with azathioprine. Most of the previous literature focused on the TPMP gene-related adverse reactions, herein, we report a case of Crohn''s disease patient with nucleoside diphosphate-linked moiety X motif 15 gene (NUDT15) variation and wild-type TPMP gene who developed toxoplasma gondii infection after azathioprine treatment.Patient concerns:A 56-year-old Crohn''s disease patient developed toxoplasma gondii infection within 2 months after the administration of azathioprine; however, he had no relevant high-risk factors.Diagnosis:Subsequent genetic testing revealed that the patient was heterozygous for NUDT15. Therefore, it was reasonable to consider that the patient''s genetic mutation resulted in reduced tolerance to azathioprine, leading to low immunity and eventually toxoplasma infection.Interventions:AZA was then discontinued; after anti-infection, antipyretic and other supportive treatments were administered, the patient''s condition gradually improved.Outcomes:The patient was followed up at 1, 3, and 6 months after discharge; fortunately, he was in good health.Conclusion:We report a case of Crohn''s disease in a patient who developed severe pneumonia caused by toxoplasma gondii infection due to the administration of AZA, with normal TPMP gene but NUDT15 gene mutation. This indicates that NUDT15 variation may contribute to severe adverse events in patients treated with azathioprine, and we suggest that NUDT15 genotype be detected before the use of azathioprine in order to provide personalized therapy and reduce side effects.     

两种免疫调节剂增强弓形虫W2b2a表位疫苗的小鼠保护作用

目的:探讨匹多莫德、沙利度胺增强弓形虫W2b2a表位疫苗刺激机体产生免疫应答和保护免疫的效果。方法:将匹多莫德、沙利度胺分别与弓形虫W2b2a表位疫苗混合肌注免疫小鼠,检测其细胞免疫和体液免疫,并观察其受到弓形虫攻击感染后的生存时间。结果:pc DNA3-W2b2a加匹多莫德组、pc DNA3-W2b2a加弗氏佐剂组、pc DNA3-W2b2a加沙利度胺组小鼠血清IFN-γ水平显著高于pc DNA3-W2b2a组(P0.05);pc DNA3-W2b2a加匹多莫德组、pc DNA3-W2b2a加弗氏佐剂组Ig G抗体水平、CD4+/CD8+比值、T细胞增殖活性显著高于pc DNA3-W2b2a组和pc DNA3-W2b2a加沙利度胺组(P0.05);小鼠攻击试验表明,免疫组小鼠存活时间明显长于对照组(P0.05)。结论:匹多莫德、沙利度胺可增强弓形虫W2b2a表位疫苗免疫小鼠的保护作用。     

刚地弓形虫RH株GRA6分子基因克隆与序列分析

目的　克隆刚地弓形虫 RH株致密颗粒抗原 (Dense granule antigen,GRA6 )分子基因 ,为研究使用重组的GRA6分子作为抗原进行弓形虫病诊断奠定基础。　方法　根据己知的 GRA6序列合成一对引物 ,抽提弓形虫速殖子m RNA,以速殖子 m RNA为模板 ,通过反转录 PCR(RT- PCR)扩增出 GRA6的 c DNA条带。目的基因 DNA条带被克隆到质粒 p UC19中形成重组质粒。对重组质粒 DNA进行纯化 ,并对目的基因片段进行核苷酸序列分析。　结果　通过RT- PCR从 m RNA中扩增出一条分子量约 70 0 bp的 DNA条带。核苷酸序列分析表明 ,GRA6分子基因的开放的阅读框全长为 6 93bp,编码 2 30个氨基酸分子 ,与已报道的 RH株 GRA6分子具有 10 0 %的同源性。　结论　本研究获得了刚地弓形虫 RH株的 GRA6分子基因 ,为今后应用此分子作为抗原诊断弓形虫病奠定了基础     

Moving towards improved vaccines for Toxoplasma gondii

Introduction: Toxoplasma gondii is an intracellular parasitic protozoan that infects almost all warm-blooded animals and humans, resulting in threats to public health and economic losses. Despite continuous research efforts, there are still very few effective strategies against toxoplasmosis. In the past few years, numerous vaccination experiments have been performed to control T. gondii infection.

Areas covered: In this review, the authors summarize the development of T. gondii vaccines with proper adjuvants, ranging from live or live-attenuated vaccines to protein vaccines, DNA vaccines, epitope vaccines and novel vaccines. They also highlight the challenges involved in the development of T. gondii vaccines, including specific impediments and shortcomings.

Expert opinion: Moving towards the development of effective vaccines against T. gondii is not only a tedious mission but also a difficult challenge. Future studies should consider new approaches and strategies for vaccine development, particularly novel vaccines and genetic adjuvants, as well as optimizing immunization protocols and evaluation criteria.