恶性疟原虫单表位单抗的建立及鉴定

目的： 研制恶性疟原虫单表位单抗。方法： 根据蛋白质结构理论， 选取能代表恶性疟原虫蛋白质抗原性的ＨＲＰ－ＩＩ肽段， 长度为６～９ 个氨基酸， 经人工合成， 通过毛细管电泳， 确定其纯度后， 对ＢＡＬＢ／ｃ小鼠进行免疫。结果： 应用脾脏埋植法和杂交瘤技术， 获得４ 株针对恶性疟原虫ＨＲＰ－ＩＩ单一表位的杂交瘤细胞。结论： 首次报告直接用合成ＨＲＰ－ＩＩ肽段制备针对该蛋白质的单表位单抗的方法获得成功。

抗小鼠PGRP-L单表位多克隆抗体的制备

目的制备抗小鼠长型肽聚糖识别蛋白(mPGRP-L)单表位多克隆抗体。方法应用生物信息学技术预测小鼠mPGRP-L分子的B细胞优势表位,人工合成抗原肽,采用MBS法将其与钥孔血蓝蛋白(KLH)偶联,免疫家兔获取mPGRP-L抗血清,采用HiTrap proteinG柱和抗原肽亲和层析柱纯化抗体,以ELISA和Western blotting进行鉴定。结果确定了位于mPGRP-L分子N端第85～104位残基的1个B细胞优势表位NH2-(C)DPHSLSPELQALISEVAQHD-COOH,合成短肽并制备KLH-肽偶联物,免疫家兔得到的抗血清效价达1∶256000。分别以HiTrapproteinG柱和抗原肽柱亲和层析纯化获得兔抗mPGRP-L单表位多克隆抗体mPGRP-Ln1和mPGRP-Ln2。纯化抗体能与重组蛋白pET-mPGRP-Ln结合,经Western blotting分析,在相对分子质量约29000处可见清晰的反应条带。结论获得抗mPGRP-L单表位多克隆抗体,为mPGRP-L分子的研究提供了工具。

多聚抗原肽免疫磁珠在制备单表位抗体中的应用

目的:建立用多聚抗原肽(multiple antigen peptides,MAP)包被磁珠制备单表位抗体的方法。方法:通过Fmoc法固相化学合成UreB的8分支单表位MAP,将其作为免疫原免疫小鼠,获得多克隆抗血清。将MAP以共价偶联的方式包被磁珠制备免疫磁珠(immunomagnetic beads,IB),通过IB从多克隆抗血清中纯化单表位抗体,荧光偏振(fluorescence polarization,FP)法鉴定抗体的特异性,SDS-PAGE鉴定抗体纯度,紫外分光光度法测定其回收率。结果:合成的MAP具有较强的免疫原性,免疫小鼠后得到的抗体滴度高达1∶12800。MAP制备免疫磁珠的最佳包被浓度为100mg/L,包被效率最高可达79%。应用MAP免疫磁珠从抗血清中纯化得到的单表位抗体,经鉴定与其他抗原表位无反应性,其纯度为95%,抗体的回收率为5.8%。结论:MAP包被的磁珠可快速分离纯化出针对某一抗原表位的特异性抗体,其性质类似单克隆抗体。这一方案在快速制备少量高特异性抗体中具有广阔的应用前景。

抗小鼠TLR2胞外段单表位抗体TSP-2对小鼠溃疡性结肠炎外周血和肠系膜淋巴结CD_4^+CD_(25)^+Foxp3^+调节T细胞表达的影响

目的观察抗小鼠Toll样受体2(Toll-like receptor 2)胞外段(mTLR2ECD)单表位抗体TSP-2对溃疡性结肠炎小鼠外周血和肠系膜淋巴结CD4+CD25+Foxp3+调节性T细胞表达的影响。方法采用硫酸葡聚糖钠(C6H7Na3O14S3,DSS)制备溃疡性结肠炎小鼠模型(UC模型),将实验小鼠随机分成四组:正常对照(NC)组、模型(MD)组、TSP-2治疗组和兔IgG治疗组。除NC组外,其他实验组均给予5%DSS造模7d后停用造模药,药物治疗组给予后续干预治疗7d。记录分析疾病活动指数(DAI),14d后处死小鼠,取结肠组织行HE染色;流式细胞仪检测外周血和肠系膜淋巴细胞中表达CD4+CD25+Foxp3+的Treg细胞的百分比率,并进行统计学分析。结果 TSP-2治疗组小鼠第9~14天DAI明显低于MD组(P<0.05);MD组外周血和肠系膜淋巴细胞CD4+CD25+Foxp3+Treg细胞占CD4+T细胞的百分率明显低于NC组(P<0.05);TSP-2治疗组外周血和肠系膜淋巴细胞CD4+CD25+Foxp3+Treg细胞占CD4+T细胞的百分率明显高于MD组(P<0.05);兔IgG治疗组与模型组比较,差异无统计学意义(P>0.05)。结论 TSP-2可以提高CD4+CD25+Foxp3+Treg数量,调节机体和肠道免疫功能,对UC发挥治疗作用。

小鼠PGRP-L分子N端基因片段的克隆与原核表达

采用RT-PCR技术,从BALB/c小鼠肝组织总RNA中扩增到长约500bp的小鼠长型肽聚糖识别蛋白(mPGRP-L)分子N端基因片段,将其克隆入pUCm-T载体构建重组质粒pmGN,DNA测序表明该基因片段长530bp,序列与GenBank中的完全一致。应用PCR技术,从重组质粒pmGN中扩增目的基因片段,插入表达质粒pET-28a构建重组表达载体pET-GN,导入大肠杆菌BL21中诱导表达目的蛋白,表达产物以Ni+-NTAagarose层析柱纯化。SDS-PAGE和Westernblot分析发现,表达产物主要以包涵体形式存在,相对分子质量约29000。ELISA表明,重组蛋白能被抗mPGRP-L分子N端单表位多克隆抗体所识别。为mPGRP-L分子的研究奠定了一定基础。

以特异性噬菌体为靶进行配基筛选的分子模建及活性鉴定

目的利用计算机辅助分子模拟技术,分析TNF模拟序列与配基的结合。方法利用计算机模建,对特异性模拟TNF的环七肽单表位克隆LCS-7(c-RRPAQSG-c)与以其为靶筛选获得的配基即TNFα结合肽LLT-18(EHMALTYPFRPP)及TNFα分子间相互结合作用进行分子模拟分析;并合成LLT-18进行ELISA鉴定与验证。结果和结论计算机模建分析显示LLT-18与TNFα表位模拟肽之间结合作用同其与TNFα分子间相互结合作用具有相似性;固相合成LLT-18短肽可与TNFα结合,证明了以噬菌体为靶进行配基筛选及分子模建预测小分子肽相互作用的可行性。

Preparation and Identification of Specific Monoclonal Antibody against Porcine Circovirus Type 2

BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.

ON THE PRODUCT OF RIESZ ALGEBRAS AND REPRESENTATION

Let { E i∶i∈I } be a family of Archimedean Riesz algebras.The product of Riesz algebras is denoted by Π i∈I E i .The main result in this paper is the following conclusion:there exists a completely regular Hausdorff space X such that Π i∈I E i is Riesz algebra isomorphic to C(X) if and only if for every i∈I there exists a completely regular Hausdorff space X i such that E i is Riesz algebra isomorphic to C(X i) .

PSMA mimotope isolated from phage displayed peptidelibrary can induce PSMA specific immune response

Prostate-specific membrane antigen (PSMA) is a cellsurface glycoprotein expressed predominantly in prostatesecretory acinar epithelium and prostate cancer cells aswell as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domainof PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5reactive phagotopes were identified. Sequence analysis ofisolated clones demonstrated that the interaction motif'VDPA/SK' has high homology to 719-725aa on PSMA.Immunohistochemical staming of the prostate cancer sam ple with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotopeisolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.

Chemical composition and surface charge properties of montmorillonite

The effects of the cell parameter and chemical composition on the surface charge properties of five kinds of different colour montmorillonites were studied. The results indicate that the surface isoelectric point（IEP） of the montmorillonite shows positive correlation with the mass fractions of Fe203 and K20, but it has little relation to the mass fractions of other chemical compositions. At around pH=6.8, the surface zeta potential of the montmorillonite shows the negative relationship with the mass fractions of Fe203 and MgO, but it does not linearly correlate to the mass fractions of other chemical compositions. Cell parameter（bo） of the montmorillonite expresses negative linear relationship with mass fractions of K2O and Na2O, so does cosinβ with mass fractions of SlOE and Fe2O3. And there is no specific relationship between bo and IEP of different montmorillonites, but there is positive correlation between cosinβ and IEP of different montmorillonite samples.

Immune Potential of a Novel Multiple-epitope Vaccine to FMDV Type Asia 1 in Guinea Pigs and Sheep

To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus (FMDV) type Asia 1 in sheep, a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized. The biologically functional molecule, the ovine IgG heavy constant region (oIgG) as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed. The recombinant proteins, RE and RE-oIgG, were expressed as a formation of inclusion bodies in E. coli. The immune potential of this vaccine regime in guinea pigs and sheep was evaluated. The results showed that IgG could significantly enhance the immune potential of antigenic epitopes. The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs, but confer complete protection in guinea pigs against virus challenge. Although the recombinant protein RE could not confer protection in the vaccinated animals, it could delay the appearance of the clinical signs and reduce the severity of disease. Inspiringly, the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination. Therefore, we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.

（Dis）Economies of Scale in Business Software Systems Development and Enhancement Projects

In the software engineering literature, it is commonly believed that economies of scale do not occur in case of software Development and Enhancement Projects （D＆EP）. Their per-unit cost does not decrease but increase with the growth of such projects product size. Thus this is diseconomies of scale that occur in them. The significance of this phenomenon results from the fact that it is commonly considered to be one of the fundamental objective causes of their low effectiveness. This is of particular significance with regard to Business Software Systems （BSS） D＆EP characterized by exceptionally low effectiveness comparing to other software D＆EP. Thus the paper aims at answering the following two questions： （1） Do economies of scale really not occur in BSS D＆EP？ （2） If economies of scale may occur in BSS D＆EP, what factors are then promoting them？ These issues classify into economics problems of software engineering research and practice.

ON THE MAXIMAL DISJOINT SYSTEM IN Riesz ALGEBRAS AND REPRESENTATION

Let E be an Archimedean Riesz algebra possessing a weak unit element e and a maximal disjoint system {e,: i∈I} in which e, is a projection element for each i. The principal band generated by eiis denoted by B(ei). The main result in this paper says that if there exists a completely regular Hausdorff space X such that E is Riesz algebra isomorphic to C(X) then for every i ∈ I there exists a completely regular Hausdorff space X, such that B(ei) is Riesz algebra isomorphic to C(Xi). Under an additional condition the inverse holds.

REMARKS ON THE REPRESENTATION THEOREM FOR RIESZ SPACES POSSESSING WEAK UNITS

The author obtains an algebraic version of the main result from his previous paper 'A characterization of Riesz spaces which are Riesz isomorphic to C(X) for some completely regular space X', and also studies the relations among some conditions used therein.

Effects of nitrogen doping on surface-enhanced Raman scattering(SERS)performance of bicrystalline TiO_2 nanofibres

In this work, we successfully synthesized bicrystalline anatase/TiO2（B） nanofibre and used it as active substrate for surface-enhanced Raman scattering （SERS） applications. The bicrystalline structured TiO2 substrates provide additional charge transfer across the anatase-TiO2（B） interface and thus enhanced activity compared to the pure single crystalline phase. With an effort to further increase the sensitivity of SERS, nitrogen element was doped into bicrystalline anatase/TiO2（B） nanofibres （N-TiO2） and higher SERS enhancement was achieved. The nitrogen content was controlled by tuning the calcination temperature of titanate precursor at 500, 600 and 700℃, respectively. The sample calcined at 600℃ （NT600） acquires the highest percentage of nitrogen element due to its open pore structure that facilitates the diffusion of nitrogen during calcination. Raman intensity depends on the amount of nitrogen doping, thus NT600 exhibited the best SERS activity. The doped nitrogen in TiO2 facilitates the charge transfer between TiO2 and probing molecules and thus suppresses the electron-hole recombination. This work provides a new perspective on the design of efficient TiO2 SERS active substrate and is expected to be valuable for adsorbate detection on semiconductor surface.

The Relevance of International Financial Reporting Standards for Small and Medium-Sized Entities （IFRS for SMEs） in Swaziland

The purpose of this study is to explore the relevance of adopting International Financial Reporting Standards （IFRS） for small and medium-sized entities （SMEs） in Swaziland. The study further seeks to determine if IFRS for SMEs can be the solution to better reporting by local SMEs. There has been a growing need for different reporting standards for small firms and big firms over the years due to the unique challenges and attributes they face especially in the area of financial reporting. This research is based on a statistical analysis of questionnaires completed by accountants from SMEs in Swaziland as well as other stakeholders who have a direct or an indirect interest in the financial statements of SMEs. These stakeholders included audit firms and the financial institutions. The sampling rate of 10% was applied across each industry to come up with a sample of 60 SMEs. Furthermore, a 100% sampling rate on the other stakeholders was applied. These were nine financial institutions and eight audit finns. The findings from the study revealed that SMEs in Swaziland are having challenges with the current reporting framework but to some extent, the framework is suitable. Most SMEs are not aware of IFRS for SMEs, therefore the accounting profession in Swaziland （SIA） has a huge role to play in training auditors and accountants in the country. Overall, IFRS for SMEs is applicable in Swaziland.

Identification of the Epitopes of Monoclonal Antibodies against P74 of Helicoverpa armigera Nucleopolyhedrovirus

P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.

表位竞争可削弱针对非优势表位的T细胞应答

目的阐明表位竞争对特异性T细胞应答强度和功能特征的影响。方法利用pSV—gag92和pSV-env203两个单表位DNA疫苗和pSV-gag／env融合基因疫苗免疫C57BL／6小鼠。免疫结束后，通过细胞内细胞因子染色的方法检测Gag92和Env203特异性T细胞反应，并比较其差异。结果pSV—gag92所活化的Gag92特异性IFN—γ＋CD8T细胞占总CD8T细胞比例[（0．41500±0．04588）％]显著高于pSV—gag／env[（0．05867±0．01964）％]。同时，我们发现pSV—gag／onv所活化的Gag92特异性TNF-α-—IFN-^γ’CD8T细胞平均荧光强度（296．70±14．08）显著低于Env203特异性TNF-（It—IFN-^γ＋CD8T细胞的平均荧光强度（818．00±49．34）。结论表位竞争能够显著降低针对非优势表位的T细胞反应频率和平均强度。

The Drainage Exchange of Stormwater Potential in Flat Area Problems

Groundwater as an alternative source still does not contribute to the water supply in area of Parit Raja because of the limitation of water availability in the ground. This lacking of groundwater could be caused by the circumstance that the top layer of soil is dominated by compacted clay around 2 meters in which its permeability is small, so the water is difficult to infiltrate the ground. The recharge well technique was designed based on the flat area problems, layer of real condition, flow water table and low infiltration rate. Resistivity soundings were made at existing wells to assess the subsurface layers. Beside that, the past records on floods event, sub surface and surface studies were collected around study area as a preliminary studies. It was presented that the study area promised good prospects to increase the capability of groundwater and contribute to the drainage system by reducing the volume of rainfall runoff using the recharge well technique.

Camrelizumab(SHR-1210),a humanized anti-PD-1 IgG4 mAb,exhibits superior anti-tumor activity and a favorable safety profile in preclinical studies

Camrelizumab is a humanized monoclonal antibody(m Ab)against human PD-1.It demonstrated a single digit nanomolar binding affinity to human and cynomolgus monkey PD-1,but no cross-reactivity to murine PD-1.It exhibited potent PD-1/PD-L1 blocking activity as well as the T cell activation in vitro.The distinct binding sites of cameralizumab were perfectly overlapped with the PD-L1 binding sites,which supported its excellent ability in blocking PD-1/PD-L1 interaction.It also significantly inhibited the growth of murine MC-38 and B16 F10 cell in humanized PD-1 transgenic mice with a superior inhibitory effect compared with the other marketed anti-PD-1 antibodies.Furthermore,camrelizumab also displayed a favorable pharmacokinetic(PK)and safety profile in cynomolgus monkeys.Besides,we showed that camrelizumab only bound to VEGFR2 with a very low affinity and did not activate VEGF pathways even at very high doses.Collectively,we provided evidence that cameralizumab was an ideal therapeutic candidate for cancer treatment,encouraging further evaluation of its efficacy/safety in the clinical setting.