弓形虫蛋白质翻译后修饰研究进展

弓形虫是一种复杂的单细胞原生动物寄生虫,通过入侵宿主细胞、分裂和诱导宿主细胞破裂等一系列的过程,在温血动物体内进行增殖。弓形虫的生物学特征受基因组、表观遗传及转录等多种因素的调控。蛋白质翻译后修饰（protein post-translational modifications,PTMs）,如磷酸化、泛素化、巴豆酰化、棕榈酰化、琥珀酰化、乙酰化、甲基化、糖基化和二羟基异丁酰化等,是弓形虫调节对细胞外刺激的反应和生命周期转变的主要机制之一。弓形虫蛋白质翻译后修饰通过改变靶蛋白的定位、结构、活性、蛋白质-蛋白质相互作用等增加蛋白质的复杂性和多样性,从而在虫体生命周期的任何时间点都可以发挥至关重要的作用。在这篇综述中,作者重点对弓形虫蛋白质翻译后修饰进行简要总结,以期为深入研究弓形虫的生物学特征奠定基础。     

桧烯抗弓形虫活性的体外评价

本研究旨在筛选具有抗弓形虫活性的萜类化合物,并进行体外活性评价。对26个萜类化合物进行体外抗弓形虫增殖作用的筛选,采用CCK-8法确定活性化合物对Vero细胞的毒性和安全浓度,Giemsa染色进一步确定对弓形虫的活性,通过细胞免疫荧光、Giemsa染色和生物化学发光法评价活性化合物对弓形虫的体外活性。结果表明,26个萜类化合物中,桧烯对弓形虫有显著的活性,桧烯对RH-2F的IC₅₀为90.76 μg·mL^-1,Giemsa染色和细胞免疫荧光的结果表明,桧烯对细胞内的两种弓形虫虫株都有显著的抗增殖作用,并且对弓形虫的入侵有极显著的抑制作用（P<0.001）。综上表明,桧烯对弓形虫的入侵和细胞内增殖具有显著的抑制作用,可作为潜在的抗弓形虫先导化合物。     

弓形虫MIC1蛋白在杆状病毒系统中的表达及活性分析

为构建可溶性表达弓形虫MIC1蛋白质的重组杆状病毒株并分析重组蛋白质的活性，通过PCR扩增弓形虫mic1基因的编码序列并连接到pFastBac 1质粒中，将重组的转移质粒转化DH10Bac感受态细胞，通过蓝白斑筛选获得重组杆粒，转染Sf9细胞后连续培养3代获得重组杆状病毒株。结果显示，Sf9细胞在感染后的第3天出现典型的细胞病变；间接免疫荧光和Western blot试验结果表明MIC1重组蛋白质在感染的Sf9细胞中成功可溶性表达；纯化的MIC1重组蛋白质不仅具有结合唾液酸乳糖的能力，同时能够刺激Balb/c小鼠产生较高水平的特异性抗体（>1∶25 600）。以上结果表明，通过杆状病毒表达系统可获得具有生物学活性的弓形虫MIC1重组蛋白质。     

3种黄芩黄酮物质体外抗弓形虫效果比较试验

为探讨黄芩中主要成分对弓形虫的抑制效果,利用MTT法对黄芩乙醇提取物、黄芩苷、黄芩素进行体外抗弓形虫作用比较试验。结果表明:3种物质均能有效地抑制弓形虫的生长繁殖,其中黄芩素的抗虫效果最好,黄芩乙醇提取物次之,黄芩苷最弱,即黄芩黄酮主要物质中,黄芩素体外抗虫作用最好。     

弓形虫代谢分泌抗原疫苗对山羊流产高发区的免疫效果试验

制备以弓形虫E/SA（代谢分泌抗原）疫苗为主,辅以衣原体疫苗,对天祝县流产高发区的299只四群适龄母山羊（对照1群）在配种前进行免疫注射效果观测。结果在产羔期结束后统计流产率较往年显著降低,免疫后的三群羊分别由上年流产率的42%、36.7%、35%降为24.39%、0%和22.88%,流产率较上年度分别降低了41.92%、100%、34.62%,未免疫的对照群却上升了52.7%（37%上升为56.5%）。经现场考察和血清学分析,排除多种因素的干扰,证明弓形虫E/SA疫苗在本次免疫中效果显著。     

成都市宠物犬弓形体血清抗体调查

本研究对成都市宠物犬中弓形体的血清抗体进行了调查。结果表明,成都市宠物犬中抗体总体阳性率达18.1%,说明成都市宠物犬中弓形体的感染普遍,应引起足够的重视。     

弓形虫重组GRA3抗原单克隆抗体的制备与鉴定

为了鉴定弓形虫特异性重组GRA3蛋白的抗原表位,以原核表达并纯化的融合蛋白GRA3免疫BALB/c小鼠,利用快速免疫法进行免疫,通过ELISA进行筛选,获得8株能够稳定分泌抗GRA3蛋白特异性单克隆抗体(McAb)的杂交瘤细胞。这8株McAbs亚类鉴定均为IgG型,特异性强,ELISA检测结果均为阳性,抗体效价均大于1∶200 000。该结果为进一步探索GRA3蛋白的结构和功能以及建立诊断方法奠定了基础。所生产的McAb可用于免疫组化和疫苗的制备,并可大大提高诊断的特异性。     

Investigation of Zoonotic Infections Among Auckland Zoo Staff: 1991–2010

Investigation was undertaken to assess the occurrence of zoonotic infection among staff at Auckland Zoological Park, New Zealand, in 1991, 2002 and 2010. Serial cross‐sectional health surveys in 1991, 2002 and 2010 comprising a health questionnaire, and serological, immunological and microbiological analysis for a range of potential zoonotic infections were performed. Laboratory results for zoo animals were also reviewed for 2004–2010 to assess the occurrence of potential zoonotic infections. Veterinary clinic, animal handler, grounds, maintenance and administrative staff participated in the surveys, with 49, 42 and 46 participants in the 1991, 2002 and 2010 surveys, respectively (29% of total zoo staff in 2010). A small number of staff reported work‐related infections, including erysipelas (1), giardiasis (1) and campylobacteriosis (1). The seroprevalence of antibodies to hepatitis A virus and Toxoplasma gondii closely reflected those in the Auckland community. No carriage of hepatitis B virus (HBV) was detected, and most of those with anti‐HBV antibodies had been vaccinated. Few staff had serological evidence of past leptospiral infection. Three veterinary clinic staff had raised Chlamydophila psittaci antibodies, all <1 : 160 indicating past exposure. Two staff (in 1991) had asymptomatic carriage of Giardia lamblia and one person (in 2010) had a dermatophyte infection. After 1991, positive tests indicating exposure to Mycobacterium tuberculosis were <10%, comparable to the general New Zealand population. Zoo animals had infections with potential zoonotic agents, including G. lamblia, Salmonella spp., Campylobacter spp. and T. gondii, although the occurrence was low. Zoonotic agents pose an occupational risk to zoo workers. While there was evidence of some zoonotic transmission at Auckland Zoo, this was uncommon and risks appear to be adequately managed under current policies and procedures. Nevertheless, ongoing assessment of risk factors is needed as environmental, human and animal disease and management factors change. Policies and procedures should be reviewed periodically in conjunction with disease monitoring results for both animals and staff to minimise zoonotic transmission.     

磺胺氯吡嗪处理弓形虫速殖子抑制消减文库的构建和初步分析

为了研究和探索磺胺氯吡嗪抗弓形虫的作用机理,构建磺胺氯吡嗪处理弓形虫的抑制性消减文库.将刚地弓形虫(Toxoplasma gondii)RH株速殖子腹腔注射(10000个/只鼠)感染昆明系小鼠,大量收集并纯化速殖子.用250 mg/mL磺胺氯吡嗪PBS溶液和PBS分别浸泡速殖子,37℃恒温箱孵育2h,分别提取虫体的总RNA和mRNA,以PBS处理的正常虫体cDNA为驱动组,以药物处理虫体cDNA为实验组,构建药物处理前后弓形虫速殖子的抑制性消减文库,对部分阳性克隆进行测序和序列分析.结果显示,提取的虫体总RNA纯度高,合成的双链cDNA经RsaⅠ酶切反应较完全;从消减文库中随机选择100个阳性克隆进行PCR扩增,其插入片段长度在200～750 bp之间,文库的重组率达93％以上;随机挑取30个阳性克隆进行测序,获得11个单一序列,Blast分析显示有7个单一有效EST序列与已知蛋白质相似性较高.结果表明,已成功构建磺胺氯吡嗪处理弓形虫速殖子的抑制性消减cDNA文库,并对部分阳性克隆进行了初步分析,为深入研究磺胺氯吡嗪的抗弓形虫机制和寻找药物作用的关键靶分子奠定了基础.