Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody

Background Thyroid peroxidase (TPO) is an important autoantigen in Hashimoto’s thyroiditis (HT), and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, purified glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO.

Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays (ELISAs). The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs.

Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid.

Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.

     

Compositions and methods for delivery of nucleic acids to hepatocytes

This patent describes the use of insect baculoviruses for the delivery of nucleic acids to human hepatocytes. Baculoviruses enter mammalian cells, particularly liver cells, but do not replicate there, making them interesting candidates for nucleic acid delivery purposes. The patent outlines glycan modifications to the virus itself to improve the efficiency of virus uptake as well as ways of making baculovirus delivered nucleic acids expressible in liver cells. A number of possible therapeutic uses are proposed, including treatment for genetic, infectious and oncogenic hepatic disease. Methods for the preparation of nucleic acid transducing baculoviruses are described.     

Eudragit S100 microparticles containing Spodoptera frugiperda nucleopolyehedrovirus: Physicochemical characterization,photostability and in vitro virus release

The aim of this study was to encapsulate the occlusion bodies (OBs) of Spodoptera frugiperda nucleopolyhedrovirus (SfNPV) in Eudragit® S100 microparticles (MPs), considering this technique as a possible alternative to protect them from deleterious environmental conditions. The MPs were prepared by oil-in-oil emulsion (O/O) solvent evaporation method. Experimental conditions were established according to a previous multi-level experimental design involving the core/polymer ratio as independent variable. The effects of these parameters on particle size and process yield were investigated observing that polymer concentration had a significant effect on particle size. After adequate conditions for MPs formation were determined, virus was encapsulated. The virus microparticles presented a particle size between 50–300 µm and concentration was 2.62 × 10⁹ OBs g^?1. Microencapsulation efficiency was 53.43% and virus release adjusted to Higuchi model suggesting diffusion as the release mechanism. Evaluated microencapsulation process protected viral particles of UV-inactivation, suggesting its potential for a biopesticide development.     

CCHFV S基因的重组杆状病毒的构建及鉴定

目的　利用杆状病毒表达系统（baculovirus expression vector systems, BEVS）构建含有克里米亚-刚果出血热病毒（Crimean-Congo hemorrhagic fever virus, CCHFV）S基因的重组杆状病毒,并在sf9昆虫细胞中表达出核衣壳蛋白（nucleoprotein, NP）。方法　将合成的CCHFV S基因定向克隆入杆状病毒转移载体pFastBac? Dual,获得重组的转移载体pFastBac? Dual-CCHFV-S。将其转化到E. coli DH10Bac?感受态细胞中,筛选得到重组杆状病毒杆粒rBV-Bacmid-S。用Cellfectin? II试剂将rBV-Bacmid-S转染入sf9昆虫细胞获得重组杆状病毒rBV-CCHFV-S。通过IFA和Western blot检测CCHFV S基因的表达。结果　构建了含有S基因的rBV-CCHFV-S,在sf9昆虫细胞中成功表达CCHFV NP。结论　利用BEVS 可以成功表达CCHFV NP,这为研究CCHFV NP的生物学特性,研制特异性的治疗药物和预防控制疫苗奠定基础。     

hTPO膜外区基因的克隆及其杆状病毒表达载体的构建

目的:探索克隆人甲状腺过氧化物酶(hTPO)膜外区基因并构建其杆状病毒表达载体。方法:PCR扩增hTPO膜外区基因,并将其先后重组入pGEM3zf(+)质粒和pFastBac1质粒,以hTPO-pFastBAC1质粒转染E.coliDH10Bac大肠杆菌,获得重组hTPO杆状病毒表达载体(hTPO-Bacmid),每一步均以PCR及酶切、基因测序等方法鉴定其正确性。结果:与预期一致,PCR扩增hTPO膜外区基因的产物为一约2.5kb的条带;hTPO-pGEM3zf(+)经EcoRⅠ+HindⅢ、EcoRⅠ+SalⅠ双酶切后均获得2.5和3.2kb条带;hTPO-pFastBAC1以EcoRⅠ+HindⅢ双酶切得到2.5和4.8kb的带,均符合预期。分别以重组hTPO-pGEM3zf(+)质粒、hTPO-pFastBAC1质粒为模板进行PCR扩增鉴定,均得到约2.5kb的目的条带;对hTPO-Bacmid进行PCR鉴定,得到约4.8kb的片段。对hTPO-pFastBAC1上下游接口测序正确无误。结论:本研究成功克隆了hTPO膜外区基因,并完成了其杆状病毒表达载体hTPO-Bacmid的构建。     

Nature of the 5′ residue in the M2 domain affects function of the human α1β1 GABAA receptor

The effects on the functional properties of the α₁β₁ GABA_A receptor when the 5′ (α₁ Val260; β₁ Ile255) hydrophobic amino acids in the second transmembrane (M2) region were changed to threonine were examined. In response to a saturating concentration of GABA, the current evoked in mutant receptors showed a decreased rate of desensitization and at equilibrium was a greater fraction of the peak current than in wild-type receptors. The half-saturation concentration of the peak current response to GABA in mutant receptors was comparable to that in wild-type receptors, but the Hill coefficient was reduced to less than one. It was concluded that the 5′ amino acids in the M2 region have a role in the conformational changes that occur within the α₁β₁ GABA_A receptor in response to GABA. Synapse 26:324–327, 1997. © 1997 Wiley-Liss, Inc.     

Heterologous expression of a substance which inhibits receptivity and calling in Helicoverpa armigera (Hübner)

The accessory glands of male moths secrete several proteins, which are known to affect post-mating behaviour in females such as calling, reduction in receptivity, rate of egg maturation and laying, sperm maintenance and release and formation of mating plug. Helicoverpa armigera (Hübner) is a polyphagous pest of numerous crops and it is widely distributed on the Indian subcontinent where it causes severe economic losses. In the present study, receptivity- and calling-inhibiting substance (RCIS), a peptide secreted from the accessory glands of male H. armigera, was sequenced, cloned and expressed in a prokaryote, Escherichia coli. RCIS is a peptide comprising 58 amino acids and had a theoretical molecular weight of 6.03 kDa. It showed 64% similarity with pheromonostatic peptide 1, identified in Helicoverpa zea (Kingan et al., 1995) but differed regarding deletion of four and one amino acids at positions 14–17 and 44, respectively, and insertion of one and five amino acids at position 38 and the terminal position of RCIS, respectively. H. armigera females injected with recombinant RCIS showed reduced receptivity and calling behaviour (in 70–80% of the treated individuals), and mating frequencies decreased by 80%. Recombinant RCIS may be employed to artificially induce non-receptivity in virgin females in order to prevent reproduction.     

人IER5基因在昆虫杆状病毒表达系统中的表达与鉴定

目的：利用昆虫杆状病毒表达系统表达早期快速反应基因5（IER5）蛋白,为后续蛋白质结晶及探索蛋白质三级结构提供线索。方法：以HeLa细胞cDNA为模板扩增出IER5基因片段,构建重组转移质粒pFastBac1-IER5;重组转移质粒经双酶切及测序鉴定后转化至感受态细胞DH10Bac,以获得重组的穿梭质粒rBacmid-IER5;将重组穿梭质粒感染Sf9细胞,待细胞出现明显病变时收集重组杆状病毒;用间接免疫荧光、SDS-PAGE及Western blot以及对表达产物进行分析鉴定。结果：重组转移质粒pFastBac1-IER5经双酶切后得到与预期相同的2条条带;重组穿梭质粒rBacmid-IER5经PCR鉴定在3 300 bp左右出现一条特异性条带;间接免疫荧光结果提示IER5蛋白在Sf9细胞中得到表达;SDS-PAGE结果显示,表达产物的相对分子质量约为48 k,Western blot结果表明表达产物能与IER5抗体特异性结合。结论：利用昆虫-杆状病毒表达系统成功表达了人IER5蛋白,获得了体外表达重组IER5蛋白的相对分子质量、溶解性等物理化学特性。     

人乳头状瘤病毒18型阳性肿瘤疫苗的构建及体外活性鉴定

目的: 制备人乳头状瘤病毒（human papilloma virus, HPV）18型阳性的肿瘤疫苗，并观察其体外活性。方法:利用昆虫杆状病毒(简称Bac to Bac)表达系统，将HPV18L1基因重组入穿梭质粒pFastBacHtb，构建HPV18 L1Htb，通过转座反应,将目的基因片段重组入杆状病毒基因