The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine

Red blood cell (RBC) transfusions are an important clinical intervention. However, RBC express hundreds of non-ABO antigens making alloimmunization a significant risk. RhD expression is the most immunologically important non-ABO antigen. Availability of RhD⁻ blood, often problematic in North America and Europe, is a significant issue in Asia and Africa where RhD⁻ blood is uncommon (<0.5% of supply). The immunocamouflage of RhD is readily accomplished by the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to the RBC membrane. To determine if RhD immunocamouflage would inhibit its immunologic recognition, an in vitro RhD-sensitized antigen presentation assay using PBMC and dendritic cells (DC) from RhD-sensitized women was used. The immunological effects of polymer grafting to an immunodominant RhD peptide, purified RhD protein and intact RhD⁺ RBC were examined via T cell proliferation and cytokine release assays. At Day 11, PEGylation significantly attenuated T cell proliferation arising from RhD peptide (∼80 → 5%), protein (36 → 0.2%) and intact RBC (33 → 1.4%). Cytokine secretion was similarly blunted following PEGylation of the purified protein or intact RBC. These data support the immunomodulatory effects of PEGylation and the potential utility of this technology in transfusion medicine - especially in situations where RhD⁻ blood is rare or in short supply.     

A Dominant Antigenic Region of the Hantaan Virus Nucleocapsid Protein is Located within a Amino-Terminal Short Stretch of Hydrophilic Residues

The nucleocapsid (N) protein of the Hantaan virus (HTNV) is a major viral antigen that induces a strong antibody response during the acute phase of infection. By immunoblot analyses of the recombinant N proteins using human sera of the hemorrhagic fever with renal syndrome (HFRS), we have confirmed previous finding by other investigators of the presence of a highly antigenic region near the amino terminus of the HTNV N protein. We have further located the antigenic region within a short stretch of hydrophilic sequences between the 26 and the 46th amino acid residues. The recombinant glutathione S-transferase fusion proteins containing this region was expressed as a soluble form in a large quantity in Escherichia coli, and purified by a single-step affinity chromatography. The recombinant antigen also showed a similar, but a weaker reactivity with human antisera to Seoul virus (SEOV), the virus most closely related to HTNV.     

不同时间脱蛋白异种骨抗原性和生物力学相关性研究

目的　研究脱脂脱蛋白处理异种骨的抗原性和生物力学强度与 30 %H2 O2 处理时间的关系 ,选择最佳处理时间 ,使骨的抗原性降至最低 ,又尽可能地保留其生物力学强度。方法　将牛松质骨脱脂脱蛋白处理 ,30 %H2 O2 浸泡时间分别为 0h ,8h ,16h和 48h。进行淋巴细胞转化实验、组织学观察和生物力学测定。结果　 30 %H2 O2 浸泡 8h组的淋巴细胞转化实验刺激指数和局部组织学反应明显低于 0h组 ,与 16h和 48h组相同 ;30 %H2 O2 浸泡 8h组的弹性模量明显低于 0h组 ,但显著高于16h和 48h组。结论　牛松质骨经 30 %H2 O2 处理 8h为最佳处理时间 ,可使抗原性降至最低 ,最大程度上保留生物力学强度。     

结核分枝杆菌38 ku蛋白的制备及其抗原性研究

目的基因克隆、表达、纯化结核分枝杆菌38ku蛋白,研究其抗原性,评价其在血清学诊断中的价值。方法2003年2月至2004年3月在中国药品生物制品检定所菌苗室以结核分枝杆菌H37Rv基因组DNA为模板,通过聚合酶链反应(PCR)方法对38ku蛋白基因进行扩增,以PET-30a( )为载体构建重组质粒,在大肠埃希菌中表达38ku蛋白,通过金属离子螯合亲和层析方法纯化重组蛋白,免疫印迹和酶联免疫吸附试验(ELISA)方法分析该重组蛋白的抗原性。结果构建了具有正确基因序列的38ku蛋白重组质粒,在大肠埃希菌BL21(DE3)中以包涵体形式表达,经过一步金属离子螯合亲和层析后得到纯度为92.7%的目的蛋白。免疫印迹试验结果表明该蛋白能与羊抗结核血清发生特异免疫结合反应。应用ELISA方法对结核血清参考品进行检测,敏感度和特异度分别为80.5%(33/41)和96.0%(25/26)。结论大肠埃希菌工程菌以包涵体的形式表达38ku蛋白,该蛋白具有良好的免疫原性,可望成为结核血清学的诊断抗原之一。     

幽门螺杆菌5种候选疫苗抗原基因的克隆、表达及抗原性的鉴定

目的:构建含人Hpylori5种候选疫苗抗原Lpp20,HspA,UreaseA,CagA,UreaseB的编码基因的重组质粒并研究其抗原性.方法:应用PCR技术从Hpylori染色体中扩增编码Lpp20,HspA,UreaseA,CagA,UreaseB的基因片段,将其T-A克隆和测序,并与GenBank公布的其他Hpylori菌株基因序列比较,再将目的基因克隆至融合表达载体pGEX-4T-1上中进行表达,用GST亲和层析对其进行纯化,纯化产物用于对29株小鼠抗Hpylori-全菌单克隆抗体(mAb)的鉴定及与Hpylori感染患者血清进行Westernblot分析.结果:扩增的Lpp20,HspA,UreaseA,CagA,UreaseB基因全长分别为528bp,351bp,675bp,855bp,1704bp(GenBank登录号分别为DQ106902,DQ141574,DQ141577,DQ141575,DQ141576),与GenBank公布的其他菌株的核酸序列的同源性在95%-99%,表达Lpp20,HspA,UreaseA,CagA,UreaseB融合蛋白的相对分子质量分别约为48000,41000,52000,60000,91000Da,29株小鼠抗Hpylori全菌mAb中针对Lpp20,HspA,UreaseA,CagA,UreaseB抗原的分别为4,5,5,1,6株,5种抗原的纯化产物均可被Hpylori感染患者血清特异性识别.结论:重组表达的Lpp20,HspA,UreaseA,CagA,UreaseB均具有较好的抗原性.     

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8％ of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1immunogenicity and antigenicity.     

猪囊尾蚴表面蛋白及其抗原性的初步测定分析

本文初步分析了猪囊尾蚴囊壁表面蛋白的特性。根据囊尾蚴漂洗液PAGE和SDS-PAGE考马斯亮蓝染色带分别显示了9条及14条蛋白区带,其中大部分与宿主骨骼肌组织的可溶性蛋白有一致的迁移率,但其中第5、7、10条蛋白带为其独有。漂洗液与囊液兔高免血清IE可出现1条沉淀弧;用漂洗液蛋白抗原对35例囊虫病人血清做ELISA,阳性率为60％。此外,对囊虫表面宿主蛋白的可能作用,做了初步探讨。     

Egg-adaptive mutations in H3N2v vaccine virus enhance egg-based production without loss of antigenicity or immunogenicity

The recently detected zoonotic H3N2 variant influenza A (H3N2v) viruses have caused 343 documented cases of human infection linked to contact with swine. An effective vaccine is needed for these viruses, which may acquire transmissibility among humans. However, viruses isolated from human cases do not replicate well in embryonated chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we sought to identify egg-adaptive mutations in surface proteins that increase the yield of candidate vaccine viruses (CVVs) in eggs while preserving their immunizing effectiveness. After serial passage of a representative H3N2v isolate (A/Indiana/08/2011), we identified several egg-adaptive combinations of HA mutations and assessed the egg-based replication, antigenicity, and immunogenicity of A/Puerto Rico/8/34 (H1N1, PR8)-based 6 + 2 reverse genetics CVVs carrying these mutations. Here we demonstrate that the respective combined HA substitutions G186₁V + N246₁K, N165₁K + G186₁V, T128₁N + N165₁K + R76₂G, and T128₁N + N165₁K + I10₂M, all identified after egg passage, enhanced the replication of the CVVs in eggs without substantially affecting their antigenicity or immunogenicity. The mutations were stable, and the mutant viruses acquired no additional substitutions during six subsequent egg passages. We found two crucial mutations, G186V, which was previously defined, and N246K, which in combination improved virus yield in eggs without significantly impacting antigenicity or immunogenicity. This combination of egg-adaptive mutations appears to most effectively generate high egg-based yields of influenza A/Indiana/08/2011-like CVVs.     

2018-2019年辽宁省甲型流感病毒流行株HA基因进化与疫苗株匹配度分析

目的分析辽宁省2018-2019年甲型流感毒血凝素(hemagglutinin,HA)基因,计算并评估流行株与疫苗株的匹配度。方法选取2018-2019年49株甲型流感毒株进行测序,对HA基因进行进化分析;研究抗原表位、糖基化位点及受体结合位点变异情况;采用Pepitope模型对流行株与疫苗匹配度进行分析。结果2018-2019年流感流行季为11月份至次年3月份,主要流行型别为新甲型H1N1亚型。经进化分析,新甲型H1N1流行株属于6B.1分支,与当年疫苗株属于同一分支;季节性H3N2毒株分布在3C.2a分支上,与2018-2019年疫苗株处于同一分支,但2019-2020年疫苗株位于3C.3a分支。部分毒株在抗原表位及受体结合位点上发生了突变。若干毒株与疫苗株相比,糖基化位点发生了增加或缺失,并未出现新的糖基化位点。使用Pepitope模型对疫苗效力从分子水平进行评估,本年度新甲型H1N1流感疫苗效力及2019-2020年疫苗株预测效力均能达到较为理想的效果。但是季节性H3N2疫苗株与本年度流行株匹配度低,计算出的疫苗效力半数以上为负值。2019-2020年H3N2亚型疫苗株效力有待进一步观察。结论2018-2019年辽宁省甲型H1N1流行株发生了一定程度的变异,H3N2亚型流行株与2018-2019年疫苗株匹配度低。应密切关注流行株的基因变异情况并及时更新疫苗株。     

可吸收性胶原止血海绵的制作工艺研究

本技术生产的可吸收性胶原止血海绵的优点：在保持天然胶原纤维机构的条件下,能有效地去除非胶原蛋白和胶原分子的非螺旋部分,具有纯度高、无免疫反应、稳定性和机械强度好的特性,技术保持领先,生产工艺相对简单,生产成本相对较低,具有一定的推广价值。