狂犬病病毒核蛋白基因在大肠杆菌中的高效表达

为获得狂犬病病毒(RV)核蛋白抗原,采用RT-PCR方法从狂犬病病毒ERA株中扩增了RV核蛋白基因,分别亚克隆到原核表达载体pET-28a(+)和pET-32a(+)中,经PCR和双酶切鉴定以及序列测定,重组质粒构建成功。将重组质粒转化到大肠杆菌BL21(DE3)中进行表达,表达的核蛋白再经Ni2+-NTA亲和层析纯化。结果显示,核蛋白在pET-32a(+)中的表达量(30.4%)高于在pET-28a(+)中的表达量(19.4%),培养物中的高纯度核蛋白产量分别为13.6和8.45 mg/L。经Western-blotting检测,不同载体表达的核蛋白均可被兔抗RV多克隆抗体特异识别,表明核蛋白具有良好的反应原性。     

牛分枝杆菌重组MPT83蛋白间接ELISA方法的建立

以纯化的牛分枝杆菌重组MPT83蛋白为包被抗原,建立了检测牛分枝杆菌抗体的间接ELISA方法。确定了间接ELISA各组分的最适反应条件:抗原包被浓度为1μg/mL,酶标二抗稀释度为1∶1600,血清稀释度为1∶60,抗原和血清、血清和二抗均在37℃反应30 min,底物在37℃显色15 min,D655 nm阴性、阳性临界值为0.5。经阻断试验、交叉试验、重复性试验,表明该方法特异性强、重复性好。用该方法对18份结核菌素试验阳性牛血清和36份结核菌素试验阴性牛血清进行检测,结果显示,阳性血清的符合率为27.8%,阴性血清的符合率为91.7%。     

The typing of group-specific component (Gc protein) in human blood stains

It is known that the typing of group-specific component (Gc protein) in human blood stains is difficult since Gc protein of the extracts of blood stains migrates more anodally to the α₁-globulin region in agar-gel immunoelectrophoresis, while Gc protein in liquid blood normally migrates to the α₂-globulin region. We have reported that the Gc protein found in the α₁-region is the result of binding of actin to Gc protein (Shinomiya, K., Kimura, H., Yoshida, K., and Shinomiya, T., J. Biochem., 92 (1982) 1163–1171, which renders it difficult to determine the Gc-phenotypes in the blood stains. On the basis of the above findings, we developed the method of phenotyping the Gc protein of human blood stains by agar-gel immunoelectrophoresis. Since the binding activity of actin to Gc protein is lost after treatment with a high concentration of guanidine HCl, the extracts of blood stains were treated with 4 M guanidine HCl to dissociate Gc protein and actin and then dialyzed to remove guanidine HCl. By this method we are able to determine the phenotypes of Gc protein in blood stains. The method we have developed is a useful tool in the forensic laboratory.     

病毒性心肌炎和扩张性心肌病中Dystrophin蛋白的表达

目的探讨病毒性心肌炎和扩张性心肌病的发病机制及相互关系,从而提高心性猝死法医学鉴定的可靠性和准确性。方法对17例对照(包括正常心脏、冠心病、高血压性心脏病等),25例病毒性心肌炎和28例扩张性心肌病的心肌组织进行改良的病理学dystrophin免疫组织化学研究。结果dystrophin蛋白在对照组,病毒性心肌炎组和扩张性心肌病组中阳性表达率分别为100%,88%,57%,三组表达差异有显著性(P<0.05),且在病毒性心肌炎和扩张性心肌病组间表达有显著差异(P<0.05),经Spearman等级相关分析呈显著负相关(r=-0.526)。结论病毒性心肌炎和扩张性心肌病心肌中细胞骨架蛋白均有破坏,且随着由病毒性心肌炎进展为扩张性心肌病,dystrophin蛋白表达逐渐降低,说明在病毒性心肌炎和扩张性心肌病的发病机制中可能与dystrophin的被破坏有关,病毒感染并破坏心肌细胞骨架蛋白并最终导致心肌细胞坏死,心功能受损,从而使病毒性心肌炎进展为扩张性心肌病。     

适配体技术研究现状与法庭科学应用展望

适配体技术是生物科技研究中的新兴发展技术之一,具有广阔的应用前景。本文从适配体技术的发展、研究方法、特性以及应用领域进行综述,并对该技术在法庭科学中的应用做出展望,以推动我国法庭科学领域对适配体技术的研究和应用。     

非洲猪瘟TaqMan实时荧光定量PCR检测方法的建立与应用

为建立检测非洲猪瘟病毒(African swine fever virus,ASFV)的快速诊断方法,本研究根据ASFV SY18株p17蛋白的编码基因D117L的保守序列设计并合成引物和探针,建立了基于ASFV p17蛋白的编码基因D117L的TaqMan荧光定量PCR检测方法,并验证其特异性、灵敏性和重复性。结果显示,本研究建立的TaqMan荧光定量PCR检测方法的C_t值与标准品在1×10^9~1×10^1copies/μL范围内呈良好的线性关系,相关系数为0.998,斜率为-3.192,检测下限为10 copies/μL,且与其他能引起相似症状的猪源病毒无交叉反应。重复性试验结果显示,组间与组内变异系数均小于1.920 7%,重复性好。此方法可用于ASF的早期诊断和ASFV快速检测。     

西尼罗病毒E蛋白的原核表达及其多克隆抗体的制备

利用PCR技术扩增西尼罗病毒(WNV)E蛋白基因并将其插入原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-WNV-E,并转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导目的蛋白表达,对诱导条件进行优化,利用His镍柱亲和层析法纯化目的蛋白,纯化后的蛋白经SDS-PAGE与Westernblot鉴定正确后,免疫BALB/c小鼠制备多克隆抗体并进行间接免疫荧光鉴定。结果显示:WNV E蛋白在大肠杆菌中以包涵体形式被成功表达,蛋白最佳诱导条件为37℃,0.8 mmol/L IPTG诱导5 h,经间接免疫荧光鉴定多克隆抗体能特异性识别WNV E蛋白。本研究成功表达并纯化了WNV E蛋白,证明该蛋白具有良好的免疫原性,为WNV抗原、抗体检测方法的建立和新型疫苗的研发及相关研究奠定了基础。     

The use of serum protein analysis in the diagnosis of fatal envenomation via Crotalus horridus (timber rattlesnake)

Deaths occurring due to rattlesnake envenomization are extremely rare and must be thoroughly investigated in the same manner as any other type of death. Our research presents the case of an adult white male who suffered a fatal timber rattlesnake (Crotalus horridus) envenomation in northwest Florida in 2018. Blood samples were taken from the decedent's heart and vasculature of the chest and sent for serum proteomic analysis. Serum proteomic analysis was utilized in order to identify proteins from timber rattlesnake (C. horridus) found within the victim's blood. The confirmation of the presence of timber rattlesnake venom within the victim's blood allows the forensic pathologist to determine the cause of death most accurately and likewise, assists with the manner of death determination. Blood samples were separated into two groups: one with the abundant endogenous proteins depleted to facilitate detection of lower abundant proteins and one undepleted. In the depleted sample, a total of 712 proteins were identified, with 47 of the proteins (6.6%) occurring originating from timber rattlesnake (C. horridus). Likewise, a total of 742 proteins were identified in the undepleted sample, with 52 of the proteins (7.0%) occurring in timber rattlesnake (C. horridus). No timber rattlesnake (C. horridus) proteins were found in control human serum.     

GFAP和VEGF在创伤性脑损伤中表达的研究进展

创伤性脑损伤是世界上导致死亡及残疾的主要原因之一。检测对中枢神经系统损伤有高度特异性和敏感性的生物标志物,可以在一定程度上反映损伤情况。胶质纤维酸性蛋白与血管内皮生长因子均可作为颅脑损伤的生物学标记物,其表达变化规律、峰值出现时间,与创伤性脑损伤的严重程度及损伤时间的推断密切相关,对于法医病理学具有重大意义。该文对胶质纤维酸性蛋白与血管内皮生长因子的分子生物学特性及其在创伤性脑损伤中的表达进行综述。     

心肌早期缺血再灌流损伤免疫组化观察

探询原癌基因c fos蛋白产物在心肌早期缺血再灌流损伤中的病理形态学改变。用SD大白鼠建立心肌早期缺血再灌流损伤模型 ,3 2只大鼠分为正常、缺血对照组与缺血再灌流组。心脏标本经HE染色及免疫组化观察。结果发现 :在缺血 3 0min再灌流 3 0min后 ,再灌流区心肌细胞有部分细胞核 ( 3 7 76%± 9 66% )呈弱阳性着色 ,而在缺血 60min再灌流 3 0min后 ,心肌细胞核 ( 4 0 3 4 %± 3 3 2 % )呈棕褐色阳性染色 ,但正常和单纯缺血组心肌细胞核未见有阳性反应 ;HE染色无明显改变。提示c fos蛋白免疫组化对显示实验性心肌早期缺血再灌流损伤有重要的价值