AIM:Novel long-acting drugs for type 2 diabetes mellitus may optimize patient compliance and glycaemic control. Exendin-4-IgG4-Fc (E4F4) is a long-acting glucagon-like peptide-1 receptor agonist. This first-in-human study investigated the safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of a single subcutaneous injection of E4F4 in healthy subjects. METHODS:This single-centre, randomized, double-blind, placebo-controlled phase 1 clinical trial included 96 subjects in 10 sequential cohorts that were provided successively higher doses of E4F4 (0.45, 0.9, 1.8, 3.15, 4.5, 6.3, 8.1, 10.35, 12.6 and 14.85 mg) or placebo (ChinaDrugTrials.org.cn: ChiCTR2100049732). The primary endpoint was safety and tolerability of E4F4. Secondary endpoints were pharmacokinetic, pharmacodynamic and immunogenicity profiles of E4F4. Safety data to day 15 after the final subject in a cohort had been dosed were reviewed before commencing the next dose level. RESULTS:E4F4 was safe and well tolerated among healthy Chinese participants in this study. There was no obvious dose-dependent relationship between frequency, severity or causality of treatment-emergent adverse events. Cmax and area under the curve of E4F4 were dose proportional over the 0.45-14.85 mg dose range. Median Tmax and t1/2 ranged from 146 to 210 h and 199 to 252 h, respectively, across E4F4 doses, with no dose-dependent trends. For the intravenous glucose tolerance test, area under the curve of glucose in plasma from time 0 to 180 min showed a dose-response relationship in the 1.8-10.35 mg dose range, with an increased response at the higher doses. CONCLUSION:E4F4 exhibited an acceptable safety profile and linear pharmacokinetics in healthy subjects. The recommended phase 2 dose is 4.5-10.35 mg once every 2 weeks.

Exendin-4 (Ex-4), one of the important glucagon-like peptide-1 receptor (GLP-1R) agonists, has proven to be an effective antidiabetic agent for type 2 diabetes (T2D).

目的 建立恶性疟原虫合子/动合子表面25蛋白(25 KDa Plasmodiumfalciparum sexual stage protein,Pfs25)的ELISA定量检测方法.方法 以双抗体夹心ELISA法为基础,建立Pfs25蛋白的ELISA定量检测方法,并对该方法进行重复性和准确性验证.用建立的方法检测不同重组菌株、CHO细胞中,以及经10L发酵罐培养菌株纯化各阶段样品中Pfs25蛋白的表达量.结果 建立的方法线性范围为0～1.6 μg/ml,R2=0.984,检测灵敏度为0.032 μg/ml,回收率为81％～119.0％,CV＜ 15％.Pfs25-Pet42a/DE3表达后的Pfs25蛋白浓度在2.03 ～7.26μg/ml;Pfs25-pGAPZαA/GS115、Pfs25-pPICZαA/GS 115和(α-Pfs25) 8-pAO815/GS 115表达后的Pfs25蛋白浓度分别为113、105和280 μg/ml;3株Pfs25/CHO细胞表达的Pfs25蛋白浓度分别为106.11、116.89和155.01 μg/ml.pPICZαA-Pfs25/GS 115菌株发酵液中,Pfs25蛋白的增加呈先快后慢的趋势,72 h后,目的蛋白增加逐渐平缓;纯化洗脱液3个峰中Pfs25蛋白浓度分别为22.67、42.12和55.27 μg/ml,流穿液中Pfs25蛋白浓度仅为0.096 μg/ml.结论 建立的Pfs25蛋白ELISA定量检测方法可用于疟疾疫苗研究中Pfs25蛋白的定量分析.

目的 制备针对恶性疟原虫环子孢子蛋白(circumsporozoite protein,CSP)NANP四肽重复序列[Asn-Ala-Asn-Pro(NANP) repeated motif]的单克隆抗体.方法 采用马来酰亚胺修饰的BSA与(NANP)5C偶联法及ADH-EDC介导的BSA与(NANP)5偶联法,将合成的NANP多肽与载体蛋白偶联,免疫BALB/c小鼠后,采用免疫后小鼠脾细胞与骨髓瘤细胞SP2/0杂交的细胞融合技术,获得分泌抗(NANP)5多肽单克隆抗体的阳性杂交瘤细胞株,间接ELISA法检测效价.通过免疫F1小鼠诱生腹水,采用50％硫酸铵沉淀法盐析纯化后,进行单克隆抗体亚类和特异性鉴定.结果 采用2种方法制备了BSA-(NANP)5偶联蛋白,以该蛋白为抗原,获得10株抗(NANP)5多肽阳性杂交瘤细胞株,效价在1:80000～1:640000之间,均为IgGl亚类,轻链类型为κ,可被(NANP)5及CSP抗原特异性识别.结论 成功制备了抗(NANP)5多肽的单克隆抗体,该抗体可在恶性疟疾疫苗的研发中发挥重要作用.

Objective To enhance the expression of β subunit of human nerve growth factor(β-NGF)in CHO cells by attenuated dihydrofolate reductase(DHFR)gene.Methods β-NGF gene was amplified from plasmid pUC18-β-NGF,and cloned into vector pMD18-T to construct cloning vector β-hNGF / pMD18-T.The cis expression element for DHFR gene,with attenuated SV40 promoter,was synthesized artificially and inserted into plasmid pBudCE4.1 to construct recombinant plasmid w-DHFR / pBudCE4.1.Recombinant plasmids β-hNGF / pMD18-T and w-DHFR / pBudCE4.1 were digested with BstBⅠ and NheⅠ,than linked and transformed to E.coli Top10'.The constructed recombinant plasmid β-hNGF / w-DHFR / pBudCE4.1 was transfected to CHO-DHFRcells,and positive clones were screened and determined for expression level of β-NGF by ELISA,and for biological activity by chick embryo dorsal root ganglion proliferation test.Results Restriction analysis and sequencing proved that recombinant plasmid β-hNGF / pMD18-T and w-DHFR / pBudCE4.1 was constructed correctly.A total of five CHO cell strains expressing β-hNGF were obtained,and the expression level of β-hNGF was 0.1 μg / μl.Obvious nerve fiber appeared in chick embryo dorsal root ganglion treated with the expressed β-hNGF.Conclusion β-hNGF with biological activity was co-expressed in CHO cells by attenuation of DHFR gene,of which the expression level increased.

目的:探讨细胞电穿孔转染的优化方法。方法:按照L9（34）正交表安排各因素水平的细胞电敏感性实验,以台盼蓝染色计数确定细胞存活率,最接近50%存活率为最优方案,根据最优方案将CHO-dhfr-细胞与DNA混匀电穿孔转染以验证正交设计结果。结果:CHO-dhfr-细胞电转染的优化参数为低离子强度Tris-Cl缓冲液、750 V/cm、50μF、电击2次、间隔1 min,各因素对转染的影响大小依次为缓冲液、电场强度、脉冲次数、电容。结论:确定了电穿孔法转染CHO-dhfr-细胞的方法,为进一步应用该细胞株表达重组蛋白打下了基础。

AIM To prepare the monoclonal antibody (mAb)against Pfs25 protein of Plasmodium falciparum, and establish the method of sandwich ELISA for detecting the Pfs25 protein. METHODS Pfs25 protein the recombinant expressed by Pichia pastoris was purified. The purified Pfs25 protein as the antigen was used to immune the BALB/c mice, The secreting specific mAb positive cell strains, which were prepared by hybridizing the Sp2/0 myeloma cell and the spleen cell from immunized mice, were detected by indirect ELISA method. The ascites of mAb were collected from immunization F1 mice, and their biological properties were identified by indirect ELISA. The anti-Pfs25 antibody was labeled by Horseradish Peroxidase (HRP), the sandwich ELISA method to detect Pfs25 protein was established based on the anti-Pfs25 mAb 4B7 and 1B4 as coating and enzyme antibody, respectively. RESULTS Three hybridoma cell lines secreting mAb against Pfs25 protein have been selected from the antibody positive hybridizing cells. The two of them have a better stability and specificity. The sandwich ELISA method detecting Pfs25 protein was established. Its detecte range was 0.07-1 mg/mL , and its sensitivity was 41.6 ng/mL. CONCLUSION The anti-Pfs25 mAb are successfully prepared and the double antibody sandwich ELISA method detecting Pfs25 protein is established. Our study lay a foundation of developing transmission-blocking malaria vaccine with Pfs25 protein as antigen.

Objective: Malaria is the mosquito-borne infectious disease and Pfs25 protein is the antigen for transmission-blocking vaccine.The aim of this study is to express the Pfs25 protein in Pichia pastoris.Methods: According to the codon-like principle for P.pastoris,the target gene was synthesized and inserted into a yeast constitutive vector pGAPZαA to construct recombinant plasmid pfs25/pGAPZαA,and then transformed the host P.pastoris GS115 genome by electroporation.The expressed product was identified by SDS-PAGE and Western blot.The recombinant P.pastoris was analyzed for genetic stability.Results: The Pfs25 protein was expressed successfully in P.pastoris,and the yeast transformants still had the foreign gene after eight passages in YPD medium.Conclusion: This study laid a foundation of future research in malaria transmission-blocking vaccine.

A rapid and sensitive method for quantitative detection of rhCNTF protein was established by double antibody sandwich ELISA.The results showed that the linearity detection range of the rhCNTF concentration is 0-25 ng/ml(r0.99),the sensitivity of the rhCNTF ELISA with a limit of detection is 0.3 ng/ml,it is of no cross reaction with other recombinant cytokines.The tested results of samples are tallied with the theoretical content data,CV(coefficient of variation) 15%.This method is of the advantage of rapid detection,the good reproducibility,highsensitivity and excellent specificity.

Objective To develop a method for determination of biologic activity and for effective quality control during preparation and production of recombinant human ciliary neurotrophic factor(rhCNTF).Methods Qualitative and semi-quantitative determinations of rhCNTF were carried out by cultures of ciliary ganalia cells and dorsal root ganglia of chicken embryo respectively.The activity of rhCNTF was determined by TF-1.CN5a.1 cell proliferation method.The biologic activity of rhCNTF in obesity was determined by bodyweight decrease test in normal mice.The four methods were investigated for experimental conditions and evaluated for advantages and shortcomings.Results All the four methods were suitable for the determination of biologic activity of rhCNTF.However,the TF-1.CN5a.1 cell proliferation method combined with bodyweight decrease test in normal mice was more effective.Conclusion The quality of rhCNTF may be controlled by TF-1.CN5a.1 cell proliferation method combined with bodyweight decrease test in normal mice.

Aim To purify recombinated GNLY which is expressed by prokaryotic expression system, and to prepare the monoclonal antibody (mAb) against it. Methods The solvable protein was purified by affinity chromatography. And employing the fusion protein GNLY immuned BALB/c mice, using conventional hybridoma technology prepared the mAb against human GNLY. Then purified and determined for titer by indirect ELISA method, for antigenic epitopes by additive ELISA, for relative affinity index by sulfocyanate elution method, and analyzed for subclass, specificity and stability. Results We got the soluble reactive fusion GNLY, and its purity and content were 95%, 0.8 g/L, respectively. Four cell strains secreting mAb against human GNLY were screened, 6C8, 9C6, 5G7and5E5. Their neutralizing titers were 1:100-1:3 200 and (0.1 - 8) x 10(-4) in supernatant and ascites. Conclusions We successfully purified the fusion protein of GNLY, and prepared the mAb against human GNLY, lying a certain foundation for its laboratory and clinical research.

Objective To prepare and identify the murine monoclonal antibody(McAb)against tumor necrosis factor-α(TNF-α)with high neutralizing activity.Methods BALB/c mice were immunized with recombinant TNF-α,based on which the McAb was prepared by hybridoma technique,then purified and determined for titer by indirect ELISA and MTT method,for antigenic epi-topes by additive ELISA,for relative affinity index by ammonium thiocyanate elution method,and analyzed for class,subclass,speci-ficity and stability.The cell strains secreting McAbs were analyzed for chromosome karyotype.Results Seven cell strains secreting McAb against TNF-αwere screened.The McAbs secreted by 6 of the 7 strains showed high neutralizing activity and stability.All the McAbs secreted by 7 strains were IgG1 withκchain,and their neutralizing titers were 1:40～1:160 and(1～3)×10-4 in supernatant and ascites respectively.The chromosome karyotypes of the 7 cell strains were consistent with those of hybridoma cells.The antigenic epitope of McAb secreted by 5H10 strain was different from those by the other 6 strains.The relative affinity indexes of McAbs se-creted by the 7 cell strains were between 0.4 and 1.5 mol/L.Conclusion The murine McAb against TNF-αwith high neutralizing activity was successfully prepared,which laid a foundation of construction of chimeric antibody against TNF-α.

Objective To optimize the condition for expression of recombinant E.coli heat-labile enterotoxin B subunit (LTB) and determine the adjuvanticity of expressed product.Methods Optimize the condition for expression of rercombinantE.coli LTB by addition of glucose to LB medium to a final concentration of 0.5% and induction at low temperature.Purify the expressed LTB by cation exchange chromatography.Immunize BALB/c mice with split influenza virus vaccine using LTB as adjuvant and determine the antibody level induced.Results After the condition for expression was optimized,the expression level of LTB increased significantly.The protein content of LTB after purification reached more than 95%.The serum and mucosal antibody levels of mice immunized with split influenza virus vaccine containing LTB as adjuvant were equivalent to even higher those containing aluminum or Freund adjuvant.Conclusion The condition for expression of recombinant E.coli LTB was successfully optimized,and the expressed LTB showed good adjuvanticity to mucosal immunization.

In order to establish anti-KDR-Fc antibody producing hybridoma cell lines,female BALB/c mice are immunized with the purified antigen of KDR-Fc, and their spleen cells are fused with SP2/0 cells to raise hybridomas. After inoculation for several times, the positive of clones are screened by indirect ELISA,and the characteristics of the antibodies are detected. Ten hybridoma cell lines are established,three of them, 1D4,4G3 and 9F10 has high biological activity,specificity and stability.

The recombinant V antigen of Yersinia pestis expressed in Escherichia coli BL21(DE3)cells was purified by way of Ni 2+ affinity chromatography. The purity was more than 90%. An alhydrogel-adsorbed vaccine of this rV antigen has been intramuscularly injected with two doses into experimental guinea pigs. It was able to induce a effective immuno-protection against subcutaneous challenge of virulent Yersinia pestis with 400 minimal lethal doses (MLD). The survival rate was 20%. These results indicate that the rV antigen is desired to act as the major component of an improved F1+V sub-unit plague vaccine.

采用分子克隆技术,将铜绿假单胞菌PA103株编码的外毒素结构域Ia(Domain Ia)的基因重组于原核表达载体pET-42b(+)上,构建了pET-EPA103蛋白表达载体.转化感受态大肠杆菌DE3.经IPTG诱导表达,初步纯化表达蛋白,用以免疫BALB/c纯系小鼠.制备小鼠脾淋巴细胞悬液,经刀豆素A(ConA)刺激后,用MTT比色法检测特异性淋巴细胞增殖反应.通过rEPA皮下注射BALB/c小鼠耳廓,诱导小鼠迟发型过敏反应(DTH).采用特异性淋巴细胞增殖反应和DTH试验来检测pET-EPA103表达蛋白所引起的小鼠细胞免疫应答水平,淋巴细胞增殖情况与DTH均可间接反映细胞免疫应答水平,进而评价重组铜绿假单胞菌外毒素(rEPA)Domain Ia蛋白片段的佐剂功效.

目的:采用酶联免疫测定法(ELISA)检测鼠神经生长因子临床用药病人的血清抗体.方法:通过直接标记鼠神经生长因子(NGF),建立了一种检测人抗鼠NGF抗体的检测方法;即双抗原夹心法检测人抗鼠NGF抗体.将此方法应用于检测临床用药病人的血清抗体,并与间接法进行了比较.结果:双抗原夹心法检测94份正常人血清,无一份假阳性,在检测临床病人血清时,亦得到了较好的结果.而间接法的非特异性则达10%.结论:双抗原夹心法方法可靠,可用于鼠源神经生长因子临床试验;血清样本检测证明临床实验病人血清抗鼠NGF抗体含量与正常人无显著差异.