目的 采用人工神经网络(artificial neural network,ANN)优化一种可获得高生物量及菌体活力大肠埃希菌(E.coli)的摇瓶培养基.方法 以葡萄糖(glucose,Glu)、酵母浸出物(yeast extract,YE)、酵母蛋白胨(yeast peptone,YP)、大豆蛋白胨(soy peptone,SP)和酵母基础氮源(yeast nitrogen base,YNB)占比为混料分量,菌体悬浮液A600(A1)、培养物湿菌重(G,g/L)和菌体活力指标值(A2,A460)为响应值,采用混料设计试验筛选对响应值具有显著影响的混料分量.以混料设计试验结果为训练和验证数据样本构建ANN模型,输入变量为混料分量且约束同混料分量上下限,输出变量为混料设计响应值.通过所构建ANN获得的优化后培养基配方和参考值,应用蒙特卡洛模拟进一步调整培养基配方,并获得E.coli摇瓶培养基配方,再对培养基配方进行10次验证试验.结果 E.coli摇瓶培养基配方:Glu26g/L、SP 26 g/L、YNB 13g/L,培养基总浓度为65 g/L.验证结果为:培养可获得A1≥14的批次几率为60％,G ≥ 77 g/L的批次几率为50％,A2≥11的批次几率为40％.培养结果数据均值与参考值均等价.结论 本研究优化获得的E.coli摇瓶培养基可培养获得高生物量及菌体活力的E.coli.

目的 建立用于检测汉逊酵母活性和活力的荧光素二乙酸酯(carboxyfluorescein diacetate,CFDA)-碘化丙啶(propidium iodide,PI)双荧光染色法.方法 通过单因素试验,优化CFDA的染色时间(5、10、20、40、60、120 min)、工作液浓度(50、100、200、300 μmol/L)及 PI 的染色时间(5、10、15、20、25 min)、工作液浓度(2、10、20、30 μmol/L).采用最适条件检测理论存活率为0、25％、50％、75％和100％的汉逊酵母样品,荧光显微镜下观察荧光信号,ImageJ软件分析灰度值;计数存活及死亡细胞个数,计算实际存活率和实际死亡率.同时分析实际灰度值、实际存活率及实际死亡率与相应理论值的相关性.采用建立的CFDA-PI双荧光染色法检测3批汉逊酵母培养物的活性及活力.结果 CFDA和PI的最佳染色时间为60和5 min,最佳工作液浓度为200和2μmol/L.不同理论存活率汉逊酵母样品的实际灰度值、实际存活率及实际死亡率与相应理论值均具有显著相关性(R2=0.998 3～0.999 2,P＜0.05).3批汉逊酵母培养物活性及活力3次检测值的CV分别为3.20％～4.03％和1.10％～2.27％.结论 成功建立了用于检测汉逊酵母活性和活力的CFDA-PI双荧光染色法,可用于相关研究中汉逊酵母活性和活力的快速检测.

目的 响应面法优化高压均质破碎重组汉逊酵母细胞的工艺条件.方法 以均质压力(A)、均质次数(B)、均质酵母质量分数(C)3个因素为影响因素,酵母细胞破碎率(Y)为响应值,应用Design-Expert软件,根据Box-Behnken中心组合试验设计方案,用响应曲面法建立二阶数学模型,确定最佳破碎工艺参数,基于二次多项式回归模型建立设计空间,并进行规模化工艺验证.结果 方差分析结果显示,模型P＜0.01,且失拟值＞0.05,表明模型与检测结果拟合较好.最终获得的操作空间为:均质压力1 125～1 200 bar,均质次数3～4次,均质酵母质量分数12％.3批重组汉逊酵母细胞发酵液中酵母细胞破碎率均达到65％以上,该值在响应值设计空间范围内.结论 响应面法优化的高压均质破碎重组汉逊酵母细胞的工艺具有较好的稳健性和适应性.

目的 评价国产阳离子交换介质Nanogel 50SP在诺如病毒样颗粒(Norovirus virus-like particles,NV-VLPs)纯化中的适用性.方法 观察并比较两种介质Nanogel 50SP和Poros 50HS在显微镜下的形态.比较Nanogel 50SP层析柱和Poros 50HS层析柱对NV-VLPs粗纯液的上样载量及两种层析柱纯化NV-VLPs粗纯液的工艺,比较Nano-gel 50SP层析柱纯化NV-VLPs粗纯液工艺的稳定性.结果 Nanogel 50SP介质形态大小较Poros 50HS更为均一.上样35个柱体积(column volume,CV)之前,Nanogel 50SP层析柱流穿液中目的蛋白纯度低于Poros 50HS层析柱,而在上样35 CV之后,Nanogel 50SP层析柱上的流穿液中目的蛋白纯度高于Poros 50HS层析柱.纯化同一批NV-VLPs粗纯液时,Nanogel 50SP层析柱收获目的蛋白的回收率稍高于Poros 50HS层析柱.3组重复纯化工艺所得目的蛋白纯度均为100％,粒径分布均符合要求,目的蛋白回收率分别为46.00％、41.98％及44.21％.结论 国产阳离子交换介质Nanogel 50SP适用于NV-VLPs的小试规模纯化,且纯化工艺有较好的稳定性.

The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Therefore, current agencies including WHO, EU, and the FDA limited the accepted amounts of residual DNA (less than 10 ng or 100 pg/dose). Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving. In this study, the detection capacity of this method was determined by comparing the detected concentration of the commercial kit and the self-designed primer/probe set after the same treatment of the extraction method. Then, a universal sample pretreatment method based on a co-precipitant was optimized. The validation results demonstrated that the method has appropriate specificity, sensitivity, accuracy, and precision according to ICH guidelines. The limit of detection and quantitation reached 3 fg/ul and 0.3 pg/reaction respectively, which satisfies the requirement of limit of residual DNA detection in biologics. Spike recovery (82.3–105.7%) showed that the proposed qPCR assay was accurate and has good extraction efficiency. Moreover, the precision of the method based on intra- and inter-assay was 0.065–0.452% and 0.471–1.312%, respectively. These results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust.

目的 用毕赤酵母表达系统表达恶性疟原虫环子孢子蛋白(CSp),便于恶性疟疾疫苗的进一步研究.方法 根据GenBank得到恶性疟原虫7G8的基因序列,选取该序列628 ～1 194位基因,以密码子最佳化为原则合成基因后构建酵母表达载体CSP/pGAPZaA,电转毕赤酵母菌PDI-GS115,获得酵母重组体.三角瓶规模表达重组菌,获得表达上清.结果 经SDS-PAGE电泳、Western blot检测表达上清,均显示有目的蛋白表达.结论 在毕赤酵母中实现了CSP基因的表达,为恶性疟疾疫苗的研发作了必要的补充.

目的:将胞内表达载体pAO815改建成分泌型表达载体,并体外构建戊型肝炎病毒ORF2128-660多拷贝重组表达质粒,进一步比较不同转化子在毕赤酵母中的表达水平,以得到高表达重组菌株.方法:利用重叠PCR技术将α-factor信号肽基因与目的基因ORF2128-660拼接后插入去磷酸化的pAO815载体,得到分泌型表达重组质粒α-ORF2128-660/pAO815(单拷贝),然后将BamH Ⅰ和BglⅡ双酶切获得的目的基因表达盒(AOX-α-ORF2128-660)插入到去磷酸化的重组质粒α-ORF2128-660/pAO815中,得到2(AOX-α-ORF2128-660)/pAO815(2拷贝),电转化毕赤酵母GS115,甲醇诱导,SDS-PAGE分析不同拷贝数转化子的表达产量,ELISA检测表达产物的生物活性.结果:α-factor信号肽基因与目的基因ORF2128-660已克隆入表达载体中,目的蛋白分泌至诱导液上清中,相对分子质量约为59 000,与阴性对照比,单拷贝、2拷贝转化子表达的产物均有生物活性但表达水平无区别.结论:成功改建成分泌型表达载体,构建了多拷贝重组表达质粒,并且成功表达了目的蛋白,为利用改造的pAO815载体表达其他蛋白奠定了基础.

Objective: To study the PEGylation of recombinant human ciliary neurotrophic factor(rhCNTF), estab-lish the preliminary separation and purification of the polyethylene glycol-modified rhCNTF, and then to detect the biological activity of PEGylation rhCNTF. Methods: Using molecular biology techniques to obtain the mutants CN10 of rhCNTF by point mutation, then to study the conditions of PEG conjugation by design of experiment. The prod-uct of PEGylation rhCNTF were purified by molecular sieve chromatography, finally the biological activity were de-tected by ELISA and the method of weight loss in normal mouse. Results: CN10 can be successfully fixed-point modified by mPEG-MAL, the weight loss in mice experiments showed that the weight increase inhibitory rate of PEGylation CN10 were up to 50% when received an intraperitoneal injection every other day, it was equivalent to inhibitory effect of rhCNTF which injected twice a day. Conclusion: After PEG modification, the biological effect of rhCNTF was significantly prolonged.

目的:Pfs25蛋白是传播阻断型疟疾疫苗重要的候选抗原,然而,I期临床研究显示该蛋白在人体内只能激发较弱的免疫应答,如何提高Pfs25蛋白质的免疫原性成为研究重点.已有的研究表明:体外通过化学偶联方法获得的Pfs25蛋白聚合物,能显著提高其免疫原性.方法:本文从基因水平,利用同尾酶的特性构建串联的Pf25基因三拷贝融合基因(Pfs25)3,每个单拷贝基因由连接臂(linker)连接,以保证每个单体Pfs25蛋白的活性不受影响.构建重组质粒(Pfs25) 3/pPICZ仅A[或(Pfs25)3/pGAPZaA]电转毕赤酵母菌,获得酵母重组体.在三角瓶规模表达重组菌并初步纯化了目的蛋白(Pfs25)3.结果:ELISA和SDS-PAGE检测表达上清,显示有弱阳性表达.获得了纯度为83％的纯品蛋白.结论:在毕赤酵母中实现了Pfs25基因的三拷贝串联表达,为恶性疟疾疫苗的研发奠定了基础.

目的 体外构建戊型肝炎病毒(HEV) ORF2128-660多拷贝重组表达质粒,以提高HEVORF2128-660在毕赤酵母中的表达水平.方法 采用PCR技术扩增目的基因ORF2128-660(E),然后将ORF2128-660(E)同义点突变为ORF2128-660,构建重组表达质粒ORF2128-660/pAO815(单拷贝),BamH Ⅰ和BglⅡ双酶切获得的目的基因表达盒(AOX-ORF2128-660)插入去磷酸化的质粒ORF2128-660/pAO815,得到2(AOX-ORF2128-660)/pAO815(2拷贝)质粒.重复上述方法依次构建3(AOX-ORF2128-660)/pAO815(3拷贝)、4(AOX-ORF2128-660)/pAO815(4拷贝)等多拷贝重组质粒.得到的多拷贝重组质粒电转化毕赤酵母菌GS115,甲醇诱导表达,SDS-PAGE分析比较不同拷贝数转化子的表达产量,ELISA检测表达产物的生物活性.结果 构建了戊型肝炎病毒(HEV) ORF2128-660多拷贝重组表达质粒,表达的目的蛋白相对分子质量约为59 000,4拷贝重组质粒表达水平高于其他拷贝数重组质粒表达水平.结论 成功构建了HEVORF2128-660多拷贝表达质粒,提高了其在毕赤酵母中的表达水平.

Objective:To explore the strategies of high-level secretory expression in order to enhance the production of Pfs25 protein in Pichia pastoris.Methods:Four main strategies were established in this study.First-ly,two kinds of recombinant Pfs25-expressing strains were constructed using the basic technology of molecular biol-ogy,and the difference between two kinds of strain was compared.The expression conditions were optimized and the best conditions being suitable for Pfs25 protein were selected.The expression medium,carbon source,pH val-ue,expression time and so on were determined.The multiple copy recombinant strains were constructed in pAO815 to enhance the expression level of Pfs25 in Pichia pastoris.The chaperone protein PDI was recombined by gene en-gineering methods into the recombinant Pfs25-expressing strains to enhance Pfs25 expression.Results:The 2 kinds of recombinant Pfs25-expressing strains were successfully constructed,and the constitutive expression was somewhat higher than inductive expression.Through comparisons of various expression conditions,the multi-copy recombi-nant strains were successfully constructed,and the Pfs25 production was higher in the 8 copy recombinant strains than in other strains.The PDI recombination enhanced the Pfs25 secretory expression in the Pfs25-expressing strains.Conclusion:The Pfs25 secretory expression in Pichia pastoris has been obviously enhanced by five folds in the optimal conditions of high-level expression strategies.

Malaria is the mosquito-borne lethal infectious disease.The pfs25 protein was proved to be the antigen for transmission-blocking vaccine.The aim of our study is to construct eukaryotic expressive vector for expression of Plasmodium falciparum transmission-blocking vaccine antigen pfs25 in CHO cell.In this study,the novel vector,in which the expression cassette was integrated with weaken SV40 promoter and dihydrofolate reductase(dhfr),was constructed and pfs25 gene was cloned into the new vector.The novel vector was designed and accomplished based on the pBudCE4.1 vector and named as pCMV-WD.It included the artificially synthetica weaken SV40 promoter,dhfr expression cassette and newly designed multiclone sites(MCS).The vector was applied especially on the CHO cell.The pfs25 gene was inserted into the pCMV-WD vector and recombinant plasmid pfs25/pCMV-WD was constructed,and then transformed into the host CHO-DHFR-cell by electroporation.Positive CHO cells expressed pfs25 gene were obtained by selection of deleting hypoxomthine,thymioline and adding zeocin antibiotics in medium.The recombinant pfs25 protein was identified by ELISA and Western Blot.The positive reaction was found in the Western Blot assay.Seven positive CHO cell lines expressed pfs25 gene were obtained,and the 0.1 mg/mL pfs25 was detected in the culture medium of 5A4 CHO cell line.The pfs25 gene was expressed successfully in the CHO cell by the eukaryotic expressive vector construction.

Objective:For the recombinant strain,with multi-copy pfs25 genes in the Pichia pastoris,study and analyze the effection of gene number on its expression level.Method:Construct the recombinant plasmid,pAO815-αpfs25,then the AOX1-αpfs25-AOX1(TT) cassette was inserted into the recombinant plasmid with single or more copy number of pfs25 gene to build multi-copy plasmids,pAO815-(αpfs25)n.Then linearized and electroporated it into GS115,screening with MD plates.Result:The recombinant strains with 1,2,3,4,5,6,7,8,10,12 and 14 copy number of pfs25 gene was obtained,the recombinant strain with eight copy number of pfs25 genes was the highest expression level.Conclusion:The recombinant strains with multiple copies of pfs25 gene was obtained successfully,no positive linear correlation was found between the expression level of Pfs25 protein and its gene number.

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.

Objective To construct a Pichia pastoris expression system with double promoter and express the zygote surface protein Pfs25 of Plasmodium falciparum.Methods Recombinant plasmids pICpfs25 with alcohol oxidase promoter 1(AOX1) and pGAPpfs25 with glyceraldehyde-3-phosphate dehydrogenase(GAP) promoter were constructed by gene recombination technique respectively and identified by restriction analysis and sequencing,then transformed to P.pastoris GS115 by electroporation to obtain recombinant P.pastoris strains ICpfs25 and GAPpfs25 respectively,based on which recombinant P.pastoris strain IC-GAP / 2pfs25 with double promoter was constructed and induced with methanol.The reactogenicity of expressed product was analyzed by Western blot,and the Pfs25 protein content in supernatants was determined by ELISA.Results The Pfs25 protein band with a relative molecular mass of about 25 000 was expressed in each of the three recombinants.However,the expression level of Pfs25 protein in the supernatant of recombinant with double promoter was 70%,which was significantly higher than those in the recombinants with a single promoter and increased gradually with the increased methanol supplement.The expressed products of various recombinants showed specific bindings to mouse anti-Pfs25 McAb 4B7.The Pfs25 protein content determined by ELISA was significantly higher in recombinant with double promoter than in those with a single promoter.Conclusion The P.pastoris expression system with double promoter was expressed,in which the expression level of target protein increased significantly.

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.

Obtain RNA from mouse anti-HBsAg hybridoma cell line,cDNA was got by RT,then the VH and VL gene of mouse anti-HBsAg was obtained for further amplification.According to the structure of VH-linker-VL,put VH and VL fragments together to form single chain Fv.Construct p26HBSc after sequencing and expressed in E.coli.Intrested protein was expressed about 30kD,It showed that it had higher affinity with HBsAg,so it is of potentially important for its humanization in the future.

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.

A primer including(Gly4Ser)3was designed,The LHSP65 gene was amplified by PCR from BCG,then ligated into expression vector pET42b(+) directly,Which was transformed into E.coli BL21(DE3) and obtained pET42-LHSP65 expression vector.It was detected by sequencing DNA and induced with IPTG.The recombinant protein is soluble,Western blotting analysis showed that the protein can react with anti-HSP65 antibody specifically.This chimeric vector is established for researching HSP65 protein.