流感病毒A/PR/8/34重配母本株高免疫原性HA蛋白制备及鉴定

制备流感病毒经典重配过程中所需的A/PR/8/34母本株血凝素蛋白(Hemagglutinin,HA)抗血清。A/PR/8/34流感病毒灭活纯化后经菠萝蛋白酶消化。5%~20%蔗糖连续密度梯度离心法纯化HA蛋白。单向免疫扩散法(Single Radial Immunodiffusion,SRID)筛选目的蛋白。SDS-PAGE法鉴定目的蛋白。纯化的A/PR/8/34 HA蛋白免疫兔子制备抗血清,血凝抑制试验(Hemoglutination Inhibition Assay,HI)测定HA抗血清效价,经SRID法鉴定HA抗血清纯度。成功制备了高免疫原性A/PR/8/34HA蛋白;兔抗血清HI滴度可达10240,SRID法鉴定为高纯度HA抗血清。制备了流感病毒经典重配中关键试剂,摸索成功整套技术方法,为我国自主研发季节性流感疫苗株奠定了基础。     

乙酰氨基半乳糖转移酶14多抗的制备及鉴定

利用生物信息学方法分析乙酰氨基半乳糖转移酶14(GALNT14)蛋白序列, 根据亲水性、抗原性、柔韧性及表面性等指标选择一段多肽序列作为抗原用于抗体制备。将该DNA片段插入pET-DsbA,构建原核表达载体pET-DsbA-GALNT14。用IPTG诱导表达蛋白,经镍柱纯化蛋白后免疫新西兰大白兔,获得GALNT14多克隆抗血清。用免疫扩散和免疫印迹检测抗体效价及特异性。结果显示,成功表达并纯化了目的蛋白。免疫动物后获得了效价较高、特异性强的GALNT14多克隆抗体,GALNT14蛋白在人乳腺癌和肾癌细胞株中均有表达。该结果为进一步研究GALNT14的功能及其在肿瘤发生发展的作用方面奠定了基础。     

人免疫缺陷病毒早期蛋白Nef的表达、纯化及免疫原性研究

目的：通过原核细胞表达人免疫缺陷病毒（HIV）Nef抗原,制备特异抗血清,为Nef抗原检测提供技术方法。方法：以HIVBotswana毒株基因组为模板,用PCR法获得Nef蛋白编码基因,将其克隆到pET30a载体中,在大肠杆菌中表达Nef融合蛋白;用纯化的融合蛋白免疫BALB／c小鼠获得抗血清,用真核表达的Nef抗原对其特异性进行分析。结果：构建的Nef融合基因在大肠杆菌中获得表达,相对分子质量约为36x103,免疫BALB／c小鼠获得针对融合蛋白的高效价抗血清,ELISA抗体滴度为1：6400;免疫荧光和Westemblot检测表明,该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论：在大肠杆菌中表达了HIVNef融合蛋白,制备了Nef融合蛋白的高效价小鼠免疫血清,该血清能特异性识别HIVNef抗原,为HlVNef抗原检测提供了技术方法。     

番茄环纹斑点病毒Gn蛋白的原核表达及多抗血清制备

通过生物信息学预测,番茄环纹斑点病毒(Tomato zonate spot virus,TZSV)Gn蛋白为跨膜蛋白,有3个跨膜结构域,抗原表位预测发现非跨膜区包含有较高的抗原指数区域,因此选择Gn蛋白的非跨膜区进行原核表达及多抗血清制备。用特异性引物,通过RT-PCR从感染TZSV的番茄中扩增出部分Gn基因片段,将获得的片段构建到原核表达载体pET-30a中,获得原核表达载体pET-30a-Gn,转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE电泳分析显示,高效诱导表达了40 kD融合蛋白。用回收纯化的融合蛋白免疫家兔,获得多抗血清。间接ELISA测定多抗血清的效价为1/16 384。利用制备好的抗血清对感染TZSV的病样进行Western blotting检测,能检测到特异性条带。结果表明该抗血清特异性良好,可用于TZSV Gn相关的免疫反应实验。     

猪FcγRIII的原核表达及多克隆抗体的制备

目的：构建猪FcγRIII 基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪 FcγRIII 抗血清。方法：从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII 原核表达载体pET-FcγRIII ,转化大肠杆菌BL21 (DE3) ,IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His 为抗原免疫小鼠,获得抗血清。Western blotting、ELISA 法鉴定获得的抗血清,ELISA 结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果：成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA 法证实多克隆抗体制备成功。结论：成功获得了猪 FcγRIII 多克隆抗体,为进一步研究猪FcγRIII 蛋白的功能奠定了基础。     

ANKRD17蛋白抗体的制备、纯化及检测

目的：制备ANKRD17（P260）蛋白的兔多克隆抗体,以与抗原相结合的方法进行抗体的纯化,并利用纯化的抗体对该蛋白进行细胞内免疫荧光检测。方法：构建表达GST—ANKRD17C端融合蛋白的质粒,在大肠杆菌中诱导表达;制备GST—ANKRD17C端抗原融合蛋白后免疫家兔,对获得的兔多克隆抗血清进行亲和纯化;纯化后的抗体经过Western blot鉴定,用于细胞免疫荧光染色检测。结果：获得较高效价的血清抗体,并对血清抗体进行了纯化;利用纯化的抗体对ANKRD17蛋白进行了细胞内免疫荧光检测,发现改蛋白定位于细胞质中。结论：制备得到的纯化抗体为研究ANKRD17蛋白的功能打下了必要的基础。     

猪FcγRⅢ的原核表达及多克隆抗体的制备

目的:构建猪FcγRIII基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪FcγRIII抗血清。方法:从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII原核表达载体pET-FcγRIII,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His为抗原免疫小鼠,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清,ELISA结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果:成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论:成功获得了猪FcγRIII多克隆抗体,为进一步研究猪FcγRIII蛋白的功能奠定了基础。     

Scytovirin在大肠杆菌中的可溶性表达

目的:获得Scytovirin(SVN)蛋白及其多克隆抗体.方法:按照NCBI上公布的SVN基因序列设计合成引物,合成SVN基因,构建pET32c-SVN原核表达重组质粒,经限制性酶切分析、DNA序列测定插入片段正确;将该重组质粒转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达;用离子交换层析法及金属亲合层析法纯化蛋白,采用Tris-Tricine系统分析;以经过纯化的蛋白为抗原免疫白兔,制备SVN多克隆抗体.结果:对表达产物进行了分离纯化,SVN纯度达到91%;用纯化的样品制备了多克隆抗体,抗血清效价为1∶102 400.结论:SVN在大肠杆菌表达系统中获得了高效可溶表达,并制备了其多克隆抗体,为进一步深入研究SVN提供了材料.     

多胺调节因子-1原核表达与多克隆抗体制备

[目的]原核表达和纯化人多胺调节因子-1(PMF-1)并以此为抗原制备多克隆抗体。[方法]PCR扩增人PMF-1编码序列并构建原核表达质粒。将此质粒转化大肠杆菌后,IPTG诱导PMF-1蛋白表达并在变性条件下用NiNTA树脂纯化。将纯化的PMF-1用作抗原免疫大耳白兔以制备抗PMF-1多克隆抗体。ELISA法检测抗体效价,免疫印迹法和细胞免疫化学法分析抗体特异性。[结果]成功构建PMF-1原核表达质粒,在大肠杆菌中IPTG可诱导该质粒高效表达PMF-1蛋白。以纯化的PMF-1蛋白为抗原免疫大耳白兔后,获得高效价的抗PMF-1抗血清(抗体效价为1∶128 000)。该抗体能与PMF-1蛋白特异性结合,并可用于PMF-1的免疫印迹和细胞免疫化学分析。[结论]成功建立了人PMF-1原核表达和纯化技术,并制备出高特异性的抗PMF-1多克隆抗体,该抗体可用于PMF-1的免疫印迹分析和细胞免疫化学分析。     

甲型H1N1流感病毒血凝素HA1在糖基工程毕赤酵母中的表达

目的:建立用糖基工程酵母制备流感血凝素的方法 ,研究其免疫原性,为酵母表达流感疫苗提供基础。方法:通过PCR的方法扩增编码H1N1流感病毒血凝素HA1(1～330 aa)的基因片段,将HA1基因克隆到表达载体pPIC9质粒上,电转化到糖基工程酵母中,甲醇诱导表达并用镍亲和层析柱纯化重组蛋白,N-糖苷酶F(PNGF)酶切分析N-糖链,Western印迹验证纯化蛋白,免疫小鼠并测定HA1诱导抗体的滴度。结果:获得HA1基因的酵母重组表达菌株,SDS-PAGE分析可见野生型GS115表达的重组HA1相对分子质量约为100×103,而糖基工程酵母GJK01表达的HA1约为60×103,PNGF酶切后相对分子质量均降至45×103左右;经Western印迹检测,这些条带均为目的蛋白条带,野生型和糖基工程酵母表达的HA1分子大小不同是由于不同的N-糖基化修饰引起的。重组HA1免疫小鼠可产生抗HA1抗体,随着抗原剂量的增加,其产生的抗体滴度相应增加;3次免疫后,4μg HA1诱导小鼠产生的抗体滴度最高。结论:利用糖基工程酵母表达制备了低糖化的流感病毒血凝素HA1,该重组蛋白可以诱导小鼠产生HA1抗体,且产生的抗体滴度具有HA1剂量依赖性。     

Titer on Chip: New Analytical Tool for Influenza Vaccine Potency Determination

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded), single radial immunodiffusion (SRID), an enzyme-linked immunosorbent assay (ELISA), and the purity adjusted bicinchoninic acid assay (paBCA). The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.     

单向放射免疫扩散法测定流感灭活疫苗血凝素含量的研究

为改进流感灭活疫苗血凝素含量的测定方法 ,将待测样品经去污剂处理后 ,用WHO标准品作对照进行免疫扩散测定 10个流感灭活疫苗样品血凝素含量。在血凝效价相同的情况下 ,疫苗中三价 (A1,A3,B)血凝素含量存在不同程度的差异。可见 ,单向放射免疫扩散法较血球凝集法更适用于流感灭活疫苗血凝素含量的测定。     

Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China

The single radial immunodiffusion (SRID) method currently used to determine the hemagglutinin (HA) content of the inactivated influenza vaccines depends on the availability of reference HA antigen and corresponding anti-serum, updated and provided annually by World Health Organization (WHO) collaborative centers. Particularly early in a pandemic outbreak, reference reagents could be the bottleneck in vaccine development and release. Therefore, other reliable tests capable of quantifying HA content could substantially shorten the time needed for vaccine formulation. Here electrophoretic separation of deglycosylated samples in conjunction with densitometry was used to quantify HA contents of H1N1 vaccine at multiple manufacturing sites. We found the overall consistency between the alternative method and traditional SRID was 88–122% in seven lots of vaccine bulks from four subtypes (types) of influenza vaccine, confirming its suitability to quantify HA content. Moreover, we used the alternative method to prepare a national HA antigen reference in China for quality control of 2009 pandemic influenza A (H1N1) vaccines prior to the arrival of the WHO SRID reference standards, subsequently confirming good agreement between both methods. The alternative method for vaccine quantification enabled the Chinese health authority to approve H1N1 vaccine 1 month earlier than otherwise possible.     

Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines

Objectives

The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed.

Results

We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months.

Conclusions

The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

     

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.     

Determination of the extraction kinetics for the quantification of polyhydroxyalkanoate monomers in mixed microbial systems

For the first time, a systematic approach was conducted to determine the key factors influencing the kinetics of hydroxyalkanote (HA) extraction in biological systems. Six mixed microbial systems where polyhydroxyalkanoate (PHA) is produced were evaluated. Experiments were carried out for full-scale and lab-scale activated sludge systems using different configurations (containing floccular or granular sludge), as well as specific PHA accumulating cultures that contain high or low intracellular PHA fractions. The overall reaction was limited by the kinetics of the PHA hydrolysis in floccular cultures, whereas in granular cultures, it was limited by the cell lysis step. The monomeric composition of the polymer also had an impact on the HA extraction rate: higher acid concentration and a longer digestion time should be employed when cells accumulate monomers with more substituents, such as hydroxy-2-methylbutyrate (H2MB) and hydroxy-2-methylvalerate (H2MV). This study optimised the method for HA extraction, which impacts the assessment of the quantity and quality of PHA biopolymers.     

流感病毒识别糖链受体分子机制的研究进展

近年来,由于流感病毒(influenza virus)不可预测的局部流行和有可能引发全球大流行,其一直是研究的热点课题之一．流感病毒表面糖蛋白血凝素(hemagglutinin,HA)特异识别宿主细胞表面的糖链受体是流感病毒感染宿主、进而复制并继续传播的生物学基础．影响流感病毒宿主特异性的两个主要因素是HA自身的变化(包括基因突变、重组、糖基化位点数量和糖基化位置的变化)和宿主细胞表面糖链受体的变化(包括糖链受体的类型、分布和分子构象的改变)等．因此准确掌握这些信息有助于人们进一步加强对流感病毒的防控．本文主要从糖组学角度概述了流感病毒识别糖链受体的分子机制,重点介绍流感病毒宿主细胞表面糖链受体的研究进展．     

四价流感病毒裂解疫苗B型血凝素含量的检测方法

目的建立能准确测定四价流感病毒裂解疫苗中B型血凝素含量(单向免疫扩散法,SRD)的检测方法。方法采用双价抗原参考品SRD法对四价流感病毒裂解疫苗中两种B型血凝素含量进行测定。将B1与B2抗原参考品按质量浓度1∶1混合制备为双价抗原参考品,双价抗原和待检样品与10%裂解剂按9∶1比例裂解30 min,分别加入到含有抗血清参考品的1.5%琼脂糖凝胶板上,每孔10μL,置20~25℃放置18~24 h,经干燥、染色、脱色,测量结果。验证双价抗原SRD法的重复性和准确性。结果双价抗原SRD法测定的血凝素含量平均值比单价抗原SRD法更接近理论配制值,故双价抗原SRD法比单价抗原SRD法能更能准确检测QIV中两种B型血凝素含量,经验证双价抗原SRD法的重复性及准确性良好。结论双价抗原SRD法提高了四价流感病毒裂解疫苗B型血凝素含量检测的准确性,为精确测定四价流感病毒裂解疫苗中B型血凝素含量提供了有效的方法和数据支持。     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

禽流感病毒血凝素蛋白的结构、功能与表达

血凝素蛋白（HA）既是一种重要吸附蛋白,介导禽流感病毒吸附和穿入宿主细胞而发挥致病作用,也是一种良好的保护性抗原,对宿主抵抗禽流感起到了决定性保护作用。研究HA蛋白对揭示禽流感病毒的致病机理和免疫防治禽流感均具有重要意义。本文重点概述了HA蛋白的结构、功能和蛋白表达方面的研究进展,并对HA蛋白在禽流感疫苗中的应用进行了探讨。