羊驼抗H5N1血凝素重链可变区-人IgGFc段嵌合抗体的制备和鉴定

本研究旨在制备羊驼抗H5N1禽流感病毒的重链抗体可变区-人Fc段嵌合体抗体制备,对所得嵌合抗体进行制备和功能鉴定,为临床应用奠定基础。用pET-22b表达载体构建抗H5N1禽流感病毒羊驼重链可变区(VHH)-人IgG1Fc嵌合基因,以包涵体形式表达VHH23-hFc嵌合抗体蛋白,采用优化的方法复性后,获得高纯度VHH23-hFc嵌合抗体,用ELISA法鉴定嵌合抗体亲和力、热稳定性和小鼠体内的半衰期。结果显示,透析复性后原核表达的抗H5N1禽流感病毒VHH23-hFc嵌合抗体亲和力为2.24×106 mol/L,具有较好免疫学活性,热稳定性也较好,小鼠体内半衰期达到35h,为下一步开展该抗体的体内外病毒中和试验奠定良好基础。     

抗H5N1禽流感病毒VHH抗体库的构建

目的:构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链可变区抗体库(VHH型抗体库),为抗H5N1的VHH抗体筛选奠定基础。方法:利用H5N1灭活疫苗免疫小羊驼,一定免疫时间后测定小羊驼外周血清中抗体中和活性,分离其外周淋巴细胞,利用RT-PCR方法得到VHH抗体片段。通过优化连接和电转化方法,将足量VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1,获得VHH抗体基因库;检测基因库库容以及多样性,并采用血凝抑制试验对噬菌体抗体库进行初步功能性鉴定。结果:利用H5N1灭活疫苗免疫小羊驼四次后,其外周血清中抗体血清抑制效价可达1∶2 560,构建的VHH抗体基因库库容可达3×108,随机挑选14个抗体基因克隆进行测序鉴定,结果显示均为独立克隆,表明所建抗体库多样性好。上述基因库经辅助噬菌体拯救后,得到抗H5N1的噬菌体VHH型抗体初级库,对初级库进行血凝抑制试验,结果呈阳性,表明初级库中存在具有潜在中和活性的抗H5N1抗体。结论:结果表明,已成功构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链抗体库,为进一步筛选抗H5N1禽流感的重链抗体打下良好基础,并为H5N1的早期临床诊断和治疗提供新的手段。     

人高致病性H5N1亚型禽流感病毒NS1蛋白多克隆抗体的制备及鉴定

目的 制备高灵敏度和高特异性的人高致病性H5N1亚型禽流感病毒NS1蛋白抗体并对其效价进行初步评估.方法 构建含有H5N1亚型禽流感病毒NS1序列的pET-28a(+)重组载体的大肠埃希菌BL21(DE3),诱导表达NS1蛋白,并经Ni-NTA色谱柱亲和层析纯化获得NS1重组蛋白,并进行SDS-PAGE和Western Blot鉴定.以纯化的蛋白为抗原免疫新西兰大白兔,获得兔抗NS1血清,亲和纯化获得多克隆抗体.应用ELISA和Western Blot检测纯化抗体的效价和特异性.结果 NS1融合蛋白得到高表达,且纯度＞90％,用该融合蛋白免疫新西兰大白兔后得到的抗NS1多克隆抗体,效价达1∶80 000,并特异性识别H5N1亚型禽流感病毒NS1蛋白.结论 获得了NS1多克隆抗体,具有较好的效价和特异性.     

H5N1禽流感病毒HA假病毒的构建及特性研究

目的 构建包膜蛋白为H5N1禽流感病毒HA蛋白的假病毒,对其生物学特性进行研究,并将其初步应用于H5N1禽流感病毒的血清检测.方法 将我国分离的高致病性H5N1禽流感病毒的HA基因插入真核表达质粒,得到pLP-HA,与假病毒构建体系的三种质粒pLP1,pLF2和pEmGFP,瞬时共转染人胚肾细胞293T,48 h收集假病毒上清,对其感染性,血凝活性进行测定,并应用于微量中和实验.同时,构建了优化HA基因的假病毒以及一株含有越南禽流感病毒HA基因的假病毒,进行比较.结果 电镜下观察到假病毒颗粒的存在;Western-Blot表明HA蛋白存在于假病毒颗粒中;HA假病毒与野生型活病毒的微量中和实验相比,两者结果具有很好的相关性.结论 成功构建了不同高致病性H5N1禽流感病毒HA蛋白的假病毒,所构建的假病毒可以应用于微量中和实验.研究发现不同禽流感病毒株HA蛋白假病毒的包装效率不同,并且真核表达优化基因并不能显著提高假病毒颗粒包装效率.     

H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析

目的:预测H5N1亚型禽流感病毒血凝素Th和B细胞相关抗原表位,并初步分析其抗原性.方法:依据近年H5N1亚型禽流感病毒流行趋势,下载得到相关HA蛋白氨基酸序列.进行生物信息学综合分析预测,获得Th和B细胞相关抗原表位,并比较其保守性和特异性.通过BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清,初步鉴定候选表位抗原性.结果:综合多项预测及空间构象模拟结果,我们获得了三条候选Th和B细胞表位,分别为HA141～155、HA206～223、HA302～316.候选表位处于H5N1亚型禽流感HA1 蛋白序列上相对保守的区域内,且与目前流行的H5N1亚型禽流感病毒HA相应区域具有较好的一致性.而不同候选表位在BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清反应中显示了不同抗体结合能力,预示了其成为功能表位的可能.结论:所筛选的表位具有成为H5N1亚型禽流感病毒HA Th和B细胞相关抗原表位的可能.本研究为深入揭示流感病毒感染与免疫机制,H5N1亚型禽流感功能表位认知及表位疫苗研究奠定了基础.     

H5N1流感病毒M1蛋白免疫制备的单克隆抗体的研究

目的 制备抗人流感病毒H5N1株M1蛋白的单克隆抗体,为流感的快速诊断和研究提供新的工具.方法 应用在大肠埃希菌中表达的人H5N1亚型禽流感病毒(A/Anhui/1/2005)株M1蛋白,以纯化的表达产物免疫BALB/c小鼠,取脾细胞与sp2/0细胞系作细胞融合后,间接ELISA法筛选阳性的杂交瘤细胞,并应用间接免疫荧光法对抗体的特异性进行鉴定.结果 获得3株能稳定分泌抗禽流感病毒M1抗原的McAb杂交瘤细胞株,交叉反应试验及间接免疫荧光检测表明,三株McAb具有型特异性.结论 用H5N1禽流感病毒M1蛋白免疫制备的单克隆抗体,具有一定的交叉反应性,可用于多种亚型甲型流感病毒的检测.     

抗人禽流感病毒H5N1人源化单链抗体噬菌体文库的构建和筛选

目的 利用抗人禽流感病毒H5N1 IgG抗体阳性的人禽流感康复患者外周血淋巴细胞,构建人源化Fv段单链抗体(seFv)噬菌体文库,并筛选与禽流感病毒相关蛋白有结合活性的scFv抗体文库.方法 提取人外周血淋巴细胞总RNA,逆转录成cDNA,以其为模板,利用家族特异性IgG基因的引物,扩增重链和轻链的可变区基因,并用合成的连接子将轻链和重链基因连接成单链抗体片段后,重组到噬菌粒载体pCANTAB5E中.将重组噬菌粒载体电转化大肠杆菌TG1,酶切和PCR鉴定抗体库的重组率,通过测定噬菌体抗体库的滴度计算抗体库的库容,用特异性禽流感病毒相关蛋白筛选表达的单链抗体.结果 构建了源于人禽流感康复患者血清的scFv抗体文库,库容为3.75×104;筛选出与禽流感病毒相关蛋白有结合活性的scFv抗体文库.结论 成功构建了抗人禽流感病毒H5N1的人源scFv噬菌体抗体库,并筛选出特异性结合人禽流感病毒相关蛋白的单链抗体,为进一步制备快速检测试剂和治疗研究提供了基础数据.     

骆驼重组SpaA-N单域抗体的制备与性质研究

目的:获得特异识别SpaA-N的单域抗体。方法:用His-SpaA-N重组抗原从新疆双峰驼单域抗体噬菌体展示文库中,筛选SpaA-N的结合子。经测序后亚克隆至pET30a并在E.coli BL21高表达,用镍离子亲和层析柱纯化。ELISA分析重组单域抗体的热稳定性,Western blot检测结合特异性。结果:经His-SpaA-N筛选富集后,筛选得到2个目的克隆。构建至pET30a,PCR和酶切鉴定目的基因大小与预计相符。SDS-PAGE显示,Mr 29 000和23 000有特异性目的条带。ELISA检测显示,抗SpaA-N的VHH对SpaA-N重组蛋白具有很好的结合活性;VHH热变性后,经室温复性均可以恢复其抗原结合活性。Western blot显示,重组VHH在Mr 66 000处可以识别丹毒丝菌中存在的表面蛋白。结论:获得了具有热稳定性和特异结合SpaA-N的单域抗体,为进一步研究spaA抗原在丹毒丝菌感染免疫中的作用提供了基础。     

慢病毒介导的H7N9禽流感病毒血凝素基因快速真核表达系统的建立

目的利用慢病毒载体构建新型H7N9禽流感病毒血凝素(HA)基因的快速真核表达体系,在人胚肾细胞(HEK293T)中表达并进行功能鉴定。方法提取H7N9禽流感病毒基因组RNA,采用反转录PCR技术扩增HA读码框全长基因,将HA基因连接pMD18-T载体构建pMD18-T-HA重组载体。设计含Kozak序列的引物从pMD18-T-HA质粒中扩增出平末端HA基因,采用Topo克隆构建表达载体pLenti-HA-V5。将表达载体转染HEK293T细胞,通过间接免疫荧光法(IFA)和Western blot法鉴定HA蛋白的表达,通过血凝实验鉴定重组蛋白的生物学活性。结果经反转录PCR获得1 683 bp的HA全长基因,完成真核表达载体的构建并表达出相对分子质量(Mr)为70 000的重组蛋白。IFA和Western blot法结果显示该蛋白与阳性血清具有良好的免疫反应,同时血凝实验证实其具有血凝活性。结论成功利用慢病毒载体建立了HA蛋白的快速真核表达系统,为研制H7N9病毒亚单位疫苗、中和表位研究、假病毒包装奠定基础。     

禽流感 HA 基因密码子优化的 DNA 疫苗构建及其免疫原性的初步研究

 目的 构建H5 N1亚型禽流感病毒株密码子优化的血凝素（HA）DNA疫苗并研究其免疫原性。方法 应用MacVector软件分析H5N1亚型禽流感病毒A/Viet Nam 1203/04株HA（H5-VN HA）基因序列，对这些序列进行密码子优化处理，人工合成优化后的H5-VN HA基因。将密码子优化H5-VN HA基因与pSW3891质粒连接，并以人组织纤维蛋白溶酶原激活剂（tPA）信号肽取代H5-VN HA信号肽，构建成表达H5-VN HA基因的重组质粒，命名为HA-VN.tPA（即密码子优化的H5-VN HA基因DNA疫苗）。以HA-VN.tPA转染293T细胞，应用蛋白质印迹法检测转染细胞中HA蛋白的表达。以HA-VN.tPA对2只新西兰兔进行DNA免疫，用ELISA方法检测分析免疫后兔血清中HA特异性抗体的产生。结果 密码子优化的H5-VN HA基因中哺乳动物细胞偏爱的密码子频率高于野生型H5-VN HA基因。蛋白质印迹法分析结果表明，HA-VN.tPA转染293T细胞后，细胞裂解液中检测出HA蛋白的表达。ELISA分析结果表明，以HA-VN.tPA免疫3次后，实验兔体内产生了较高水平的HA特异性抗体（血清中平台期抗体滴度达1:13 500）。结论 成功构建了H5N1亚型禽流感病毒密码子优化的HA 基因DNA疫苗，该疫苗可引导免疫后宿主体内产生高滴度特异性抗体。     

Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays

From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children's sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.     

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.     

H5N1 receptor specificity as a factor in pandemic risk

The high pathogenicity of H5N1 viruses in sporadic infections of humans has raised concerns for its potential to acquire the ability to transmit between humans and emerge as a highly pathogenic pandemic virus. Because avian and human influenza viruses differ in their specificity for recognition of their host cell receptors, receptor specificity represents one barrier for efficient transmission of avian viruses in human hosts. Over the last century, each influenza virus pandemic has coincided with the emergence of virus with an immunologically distinct hemagglutinin exhibiting a ‘human-type’ receptor specificity, distinct from that of viruses with the same hemagglutinin circulating in zoonotic species. Recent studies suggest that it is possible for H5N1 to acquire human type receptor specificity, but this has not occurred in nature. This review covers what is known about the molecular basis for the switch between avian and human-type receptor specificity for influenza viruses that have successfully adapted to man, the potential for H5N1 to evolve to human-type receptor specificity and its relevance to pandemic risk.     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.     

Evaluation of a rapid test for detection of H5N1 avian influenza virus

The performance of H5 Dot ELISA, a rapid test for detection of avian H5N1 influenza virus, was evaluated using 30 H5N1 strains belonging to 10 major genetic groups of H5N1 influenza virus, 14 strains of non-H5N1 influenza virus and 652 field samples collected from healthy and diseased chickens from markets and poultry farms. The detection limit of the test for all 30 strains of H5N1 virus was < or = 0.1 hemagglutinin (HA) units and the test yielded a negative result when tested against 100 HA units of the non-H5N1 viruses. The test gave a positive result for 87 of the 106 poultry samples from which H5N1 virus was isolated by culture and 3 of 546 culture-negative poultry samples. Compared with virus culture, the overall prediction rate of the test was determined to be 96.6%; the positive prediction rate was 96.7% and negative prediction rate, 96.6%. The false positive rate was 0.5% and false negative rate 17.9%. Considering that the test is also convenient to use, it was concluded that H5 Dot ELISA is suitable for field use in the investigation of H5N1 influenza outbreaks and surveillance in poultry.