用M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达HIV-1 gp41抗原表位

目的:探讨M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达融合蛋白的可行性。方法:以纯化的HIV-1感染者血清多克隆抗体为配体,在噬菌体展示随机十二肽库中进行生物淘洗,经ELISA鉴定阳性克隆,DNA测序,确定优势表位。将优势表位及两个优势表位的串联体分别与M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域连接,克隆入PQE30载体进行蛋白表达,再以表达蛋白为抗原检测HIV-1感染者血清中的抗体。结果:成功筛选到位于HIV-1 gp41蛋白上的3组优势抗原表位(HGPKDAETTAIW;AAFKDNQLLRIW;AAFKDNQLTRIW),3组优势表位及表位串联体(YGPKDAETTAIW-GGGS-SCSAKFTCTTQI)在PQE30载体中实现可溶性融合表达。重组蛋白具有良好的抗原性,能与不同的HIV-1抗体阳性血清呈特异反应。结论:用M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达HIV-1 gp41抗原表位是可行的。     

用M13噬菌体P H蛋白Ⅰ-Ⅱ结构域表达HIV-1 gp41抗原表位

目的：探讨M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达融合蛋白的可行性.方法：以纯化的HIV-1感染者血清多克隆抗体为配体,在噬菌体展示随机十二肽库中进行生物淘洗,经ELISA鉴定阳性克隆,DNA测序,确定优势表位.将优势表位及两个优势表位的串联体分别与M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域连接,克隆入PQE30载体进行蛋白表达,再以表达蛋白为抗原检测HIV-1感染者血清中的抗体.结果：成功筛选到位于HIV-1 gp41蛋白上的3组优势抗原表位（HGPKDAETTAIW;AAFKDNQLLRIW;AAFKDNQLTRIW）,3组优势表位及表位串联体（YGPKDAETTAIW-GGGS-SCSAKFTCTTQI）在PQE30载体中实现可溶性融合表达.重组蛋白具有良好的抗原性,能与不同的HIV-1抗体阳性血清呈特异反应.结论：用M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达HIV-1 gp41抗原表位是可行的.     

人乳头瘤病毒重组质粒的构建及蛋白表达

目的 构建重组原核表达质粒获得人乳头瘤病毒18型L1片段(HPVl8LI)活性蛋白。为进一步研制人乳头瘤病毒18型(HPV18)基因工程疫苗打下基础。方法 以重组质粒(pBR322-HPV18)为模板，利用PCR方法扩增HPV18L1DNA片段。将HPV18L1DNA与质粒(pUC19)重组构建重组质粒(pUC19-HPV18L1)。用酶切电泳验证重组结果的正确性；并通过测序检查质粒重组后序列有无变化。再利用质粒(pQE32)做表达载体构建重组质粒(pQE32-HPV18L1)，并用酶切电泳验证。将重组质粒pQE32-HPV18L1转入工程菌(M15)。用酶切电泳验证重组工程菌(pQE32-HPV18L1／M15)正确性。利用SDS-聚丙酰胺凝胶电泳(SDS-PAGE)检测HPV18L1目的蛋白。采用不同浓度异丙基-β-D-半乳糖苷(IPTG)、不同诱导时间来优化HPV18L1目的蛋白表达条件。利用Western印迹检测蛋白质(Western blot)鉴定HPV18L1目的蛋白的特异性。结果PCR扩增DNA片段约为1．7Kb。与预期结果相同。克隆重组质粒pUC19-HPV18L1酶切后显示的酶切图谱与预期相同。而且测序验证插入片段全序列无改变。表达重组质粒pQE32-HPVl8L1酶切图谱亦与预期相同。蛋白表达条件优化结果为1mmol／LIPTG诱导4h后，获得HPV18L1最佳蛋白表达量：SDS-PAGE显示，约63KD处可见目的蛋白带，与预期结果一致。Westem blot鉴定，在约63KD处可见目的条带，与预期结果一致。结论 成功构建表达重组质粒pQE32-HPV18L1并能表达HPV18L1目的蛋白。     

重组多价人精子表位肽的原核表达及其条件优化

目的构建重组多价人精子表位肽原核表达质粒,优化其在大肠杆菌中的表达条件。方法用化学合成法合成多价人精子表位肽基因。该基因经PCR扩增,BamHI和XhoI双酶切后,克隆至GST融合表达载体PGEX-4T-1获得重组质粒。将该重组质粒转化E.coli BL21(DE3),经IPTG诱导表达。利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统,观察在摇瓶发酵条件下改变培养基组成、诱导温度、诱导时机、诱导剂浓度和诱导时间等条件对GST-多价人精子表位肽融合蛋白表达量的影响。结果构建了重组多价人精子表位肽原核表达载体。在摇瓶实验中,优化的表达条件为:TB培养基,诱导温度37℃,在菌体对数生长的中后期进行诱导,IPTG诱导终浓度0.05 mmol/L,诱导时间5 h。优化后GST-多价人精子表位肽融合蛋白的表达量占菌体总蛋白的30.2%,且主要以可溶形式表达。结论成功构建重组多价人精子表位肽原核表达质粒,重组表达载体在E.coli BL21(DE3)内主要表达可溶形式的GST-多价人精子表位肽融合蛋白,在优化条件下可获得较高的表达量。     

SARS冠状病毒多抗原表位融合蛋白的表达及其诊断应用

目的 制备SARS冠状病毒(SARS-CoV)多抗原表位融合蛋白,建立一种可特异性诊断SARS的血清学方法,用于SARS的明确诊断和流行病学调查研究.方法 设计含有SARS冠状病毒S、M和N蛋白优势抗原表位区的多表位融合蛋白SMN,利用大肠杆菌表达SMN蛋白,并作为包被抗原建立ELISA方法,用于SARS患者血清检测.结果 设计出包含S603-635 M2-31、N362-412表位区的多表位融合蛋白SMN,并在大肠杆菌中成功表达.以SMN蛋白为包被抗原,ELISA检测74份SARS患者血清全部为阳性,另44份非SARS-CoV感染的发热病人血清和62份健康人血清均为阴性.结论 SARS-CoV多抗原表位融合蛋白SMN在大肠杆菌中成功表达,以SMN为检测抗原建立的ELISA方法为SARS血清学诊断奠定了基础.     

戊型肝炎病毒结构蛋白基因的真核表达

目的使用酵母表达系统表达戊型肝炎病毒结构蛋白基因。方法构建含戊型肝炎病毒(HEV)结构蛋白嵌合基因HEVORF23(由HEVORF2C端800bp片段与ORF3全基因组成)的重组表达质粒pPICZαA_HEVORF23,并通过电转移的方法将该基因整合入甲醇营养型酵母的基因组DNA中,利用甲醇进行诱导表达,表达产物经纯化后,进行Western印迹鉴定。结果pH6.0环境下诱导培养72h后,在细胞沉淀中获得一个分子量大约为69kDa的可溶性目的蛋白,纯化蛋白经Western印迹检测后发现能与戊型肝炎患者血清发生较强的特异性免疫反应。结论采用酵母表达系统诱导表达可以获得抗原性较强的HEV重组表达抗原,为进一步开发研制有效的诊断抗原和疫苗奠定了基础。     

HPV 18型E6蛋白抗原表位预测与分析

目的 用生物信息学方法分析预测HPV 18型E6蛋白的B细胞和T细胞表位,为宫颈癌免疫治疗的疫苗及药物开发提供参考靶点。方法 基于NCBI中HPV18型E6蛋白的氨基酸序列,利用DNA star软件预测其二级结构、亲水性、表面可及性及柔韧性;利用ABCpred软件预测出其B细胞表位;综合使用CTLPred和NetMHC 4.0软件分析其CTL表位;ProPred和NetMHCIIpan 3.1软件预测其Th表位。结果 HPV 18型E6蛋白二级结构中α螺旋和β转角较多且分布较为均匀;预测出了4个B细胞表位,它们大多与亲水性强、表面可及性好、柔韧性好的蛋白区域重叠,易于结合抗体;E6蛋白具有丰富的细胞毒性和辅助性T细胞表位。结论 HPV 18型E6蛋白作为宫颈癌免疫治疗的理想靶点具有同时诱导特异性体液免疫和细胞免疫的潜能,通过对其B/T细胞抗原表位的预测为免疫治疗相关疫苗及药物抗原肽的选择提供了理论依据。     

麻疹病毒血凝素蛋白抗原表位的预测与分析

目的 探讨麻疹病毒(MeV)流行株血凝素蛋白(H)抗原表位上氨基酸(aa)变异对病毒抗原性的可能影响。方法 利用生物信息学软件预测MeV H蛋白上B细胞线性表位,设计并合成来源于疫苗株和流行株表位以及同一区域非表位上的多肽对。间接ELISA法检测合成多肽的免疫原性,并制备多肽免疫血清。采用交叉ELISA法分析两条多肽间的抗原性差异,计算抗原比。结果 合成的多肽均能与MeV免疫血清结合,其中设计在表位区的多肽对CW23/CW22(273～282 aa)结合能力最强,而非表位区多肽对CW150/CW151(418～427 aa)结合能力最弱。多肽对中来源不同两条多肽间抗原性差异较大,其中CW23(疫苗株来源)与CW22(流行株来源)间抗原比为16,CW123(疫苗株来源)与CW124(流行株来源)(236～246 aa)间的抗原比为2.877±0.583。非表位多肽对中,CW125与CW126(356～364 aa)间抗原比为1.631±0.481,而CW150与CW151间抗原比为10.367±1.617。结论 麻疹流行株上仍存在保守的抗原表位,但预测的抗原表位及非表位区上的部分aa变异导致疫苗株与流行株间抗原性存在差异。     

重组诺如病毒GII 4型衣壳蛋白的表达、纯化及鉴定

目的通过基因重组方式获取高浓度重组诺如病毒GⅡ4型衣壳蛋白。方法将诺如病毒GII 4型衣壳蛋白基因片段插入pHT A载体中,测序鉴定正确后,将重组质粒转化到MAX Efficiency~ DH10Bac~(TM)感受态细胞,表达目的蛋白。重组蛋白用Ni-NTA His蛋白亲和柱纯化,用诺如病毒检测试剂盒进行鉴定。结果 SDSPAGE结果表明成功表达了分子量约为60 kD的重组蛋白,经诺如病毒检测试剂盒鉴定,表达产物为重组诺如病毒衣壳蛋白,并且具有较好的免疫原性。结论构建了诺如病毒GII 4型衣壳蛋白表达载体,并获得重组诺如病毒GII 4型衣壳蛋白。     

丙型肝炎病毒核心区多肽的抗原表位分析和原核表达

目的 分析HCV核心区多肽的抗原表位,选择具有优势抗原表位的多肽用原核表达载体表达,并获得该表达载体的稳定表达菌株. 方法 根据软件分析选取带有优势抗原表位的HCV核心区多肽,找出对应DNA序列,逆转录PCR扩增目的基因,克隆到原核表达载体中进行诱导表达.表达的目的蛋白用His Bind Columns纯化,用ELISA法检测其免疫活性. 结果 获得了序列正确的HCV-core基因和表达良好的该基因原核表达载体pET-28a-core,该表达载体表达的目的蛋白可被很好地纯化并具有良好的HCV抗原活性. 结论 成功地重组了HCV核心区蛋白,获得了HCV抗原性良好的HCV核心区抗原.     

HIV-1 gp41基因片段亚型多样性研究

〔目的〕应用HIV-1 env gp41基因分型技术进行出入境HIV感染者HIV-1亚型多样性研究。〔方法〕采用套式逆转录PCR方法对env gp41和gp120(C2-V3区)进行基因扩增、测序、亚型对比分析,以及应用威斯康星GCG公司软件进行系统树分析和用美国Los Alamos国家实验室的HIV-Blast软件进行序列的亚型鉴定。〔结果〕对22份样本的gp41和gp120(C2-V3区)的基因亚型进行对比分析,结果表明gp41和gp120(C2-V3区)在亚型上具有一致性。对65例来自中国、非洲、欧洲、美洲和东南亚的出入境HIV-1阳性者进行gp41扩增基因亚型分析,发现有A、B、C、D、F1、G亚型和重组亚型AC、01_AE、02_AG、07_BC、08_BC、06_cpx、18_cpx共计13种亚型,其中重组株占33.8%(22/65)。中国籍出入境感染者包括了A、B、C、D、F1、G亚型和重组亚型01_AE、02_AG、07_BC、08_BC在内的10种亚型,其中重组株占25%(7/28)。〔结论〕用gp41替代gp120(C2-V3区)进行HIV-1亚型分析较为理想。出入境人群中HIV感染者感染的HIV毒株几乎包括了全球流行的所有亚型,多种国内少见的亚型已经通过国际旅行者传入国内。     

Molecular investigations into vaginal immunization with HIV gp41 antigenic construct H4A in a quick release solid dosage form

A robust vaginal immune response is considered essential for an effective prophylactic vaccine that prevents transmission of HIV and other sexually acquired diseases. Considerable attention has recently focused on the potential of vaginally administered vaccines as a means to induce such local immunity. However, the potential for vaccination at this site remains in doubt as the vaginal mucosa is generally considered to have low immune inductive potential. In the current study, we explored for the first time the use of a quick release, freeze-dried, solid dosage system for practical vaginal administration of a protein antigen. These solid dosage forms overcome the common problem associated with leakage and poor retention of vaginally administered antigen solutions. Mice were immunized vaginally with H4A, an HIV gp41 envelope based recombinant protein, using quick release, freeze-dried solid rods, and the immune responses compared to a control group immunized via subcutaneous H4A injection. Vaginally immunized mice failed to elicit robust immune responses. Our detailed investigations, involving cytokine analysis, the stability of H4A in mouse cervicovaginal lavage, and elucidation of the state of H4A protein in the immediate-release dosage form, revealed that antigen instability in vaginal fluid, the state of the antigen in the dosage form, and the cytokine profile induced are all likely to have contributed to the observed lack of immunogenicity. These are important factors affecting vaginal immunization and provide a rational basis for explaining the typically poor and variable elicitation of immunity at this site, despite the presence of immune responsive cells within the vaginal mucosae. In future mucosal vaccine studies, a more explicit focus on antigen stability in the dosage form and the immune potential of available antigen-responsive cells is recommended.     

Development of a DNA vaccine expressing a secreted HIV-1 gp41 ectodomain that includes the membrane-proximal external region

A limited number of sites on the HIV-1 Envelope protein are vulnerable to broadly neutralizing antibodies (bnAbs). One of these sites, the membrane proximal external region (MPER), is located at the C-terminus of the gp41 ectodomain (gp41_ecto). This highly conserved sequence is bound by several well-characterized bnAbs. Efforts to produce a gp41 immunogen are in part hampered by the MPER’s hydrophobicity and propensity to induce aggregation. We sought to produce a DNA vaccine expressing a gp41_ecto that is both secreted from mammalian cells and maintains binding by bnAbs to the MPER. Through in silico analysis, we predicted regions of gp41_ecto that could induce aggregation and possibly hinder secretion. We generated deletion mutants of gp41_ecto and tested their ability to be secreted by mammalian cells. Upon deletion of regions in either the fusion peptide (FP) or MPER, secretion of the gp41_ecto increased. In an effort to both augment secretion and maintain binding by bnAbs, we developed constructs with the FP deletion and introduced point mutations in the MPER. Two constructs (gp41 ΔFP and gp41 ΔFP+I682E) maintained binding by gp41 MPER-specific bnAbs (4E10, Z13e1 and 10E8). These were evaluated as DNA vaccines in a mouse model. Both vaccines proved to be immunogenic and appeared to elicit some MPER-specific antibodies that bound gp41 ectodomain-derived proteins but not short peptides spanning the MPER. No neutralizing capacity was detected against a clade C virus containing a homologous MPER.     

Role of lipid structure in the humoral immune response in mice to covalent lipid–peptides from the membrane proximal region of HIV-1 gp41

The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 × 10⁴) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 × 10² IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine.     

gp85 protein vaccine adjuvanted with silica nanoparticles against ALV-J in chickens

This study focused on the effect of silica nanoparticles as adjuvant for vaccine applications comprised of gp85, a dominating structural protein of J Subgroup Avian Leukosis Virus (ALV-J), and which was evaluated by comparing with the responsiveness induced by that emulsified in Freund adjuvant. Thirty-six chickens were inoculated twice with gp85 adjuvanted with the silica nanoparticles or Freund’s adjuvant at the 2nd and 3rd week old. Two weeks later, the inoculated chickens were challenged with a 10^2.2 50% tissue culture infective dose (TCID50) of ALV-J. The blood samples were collected weekly to detect the serum antibodies and viremia. Results showed that positive serum antibodies (S/P value > 0.6) against gp85 emerged at the third week in the inoculated chickens, while the antibodies level persisted longer in silica nanoparticles adjuvanted-group to Freund’s adjuvanted-group. Furthermore, viremia in silica nanoparticles adjuvanted-group was recovered more quickly compared with Freund’s adjuvanted-group. Hence our study revealed that silica nanoparticles can effectively improve the protection of gp85 vaccine against ALV-J and present a better performance than Freund’s adjuvant.     

巨细胞病毒gp52蛋白表达与应用

目的 构建表达巨细胞病毒(CMV)gp52蛋白抗原的重组质粒及工程菌,获取纯化的gp52蛋白抗原.方法 采用PCR技术,克隆表达CMV gp52重组蛋白,利用产物建立IgM捕获ELISA方法鉴定其抗原性及实用性.结果 表达纯化的gp52蛋白纯度＞95%,经酶标记建立IgM捕获ELISA方法,检测35份抗巨细胞病毒IgM阳性血清和35份阴性血清,用酶标记gp52蛋白建立的捕获ELISA法阳性检出率97.1%,阴性检出率100%,与意大利SORIN公司试剂盒检测结果比较,差异无统计学意义(P＞0.05);其中1份CMV-IgM阳性血清1:16稀释后仍能与抗原反应,表明gp52蛋白抗原表位有较好的抗原特异性.结论 高效表达纯化的gp52蛋白抗原性强,利用其建立的IgM捕获ELISA方法,可用于检测巨细胞病毒抗体.     

Prime boost vaccination approaches with different conjugates of a new HIV-1 gp41 epitope encompassing the membrane proximal external region induce neutralizing antibodies in mice

Peptide mimics of epitopes for pathogen-specific antibodies present in patient sera can be selected based on the phage display technology. Such mimotopes potentially represent vaccine candidates in case they are able to induce neutralizing antibodies upon vaccination. Here we analyze the immunogenicity of different conjugates of epitope EC26-2A4 localizing to the membrane proximal external region (MPER) of the HIV-1 transmembrane protein gp41. The EC26-2A4 epitope, which overlaps with that of the broadly neutralizing monoclonal antibody (mAb) 2F5, was coupled to sequential oligopeptide carriers (SOC) or to palmitoyl acid for better immunogenicity. Upon prime-boost immunizations of mice, the peptide conjugates induced EC26-2A4 specific antibodies in all settings and mice sera neutralized HIV-1SF162.LS in standardized neutralization assays. Thus, the EC26-2A4 MPER epitope represents a promising vaccine candidate for further analysis in larger animals with respect to the breadth of the neutralizing antibodies induced.     

Cooperative effects of immune enhancer TPPPS and different adjuvants on antibody responses induced by recombinant ALV-J gp85 subunit vaccines in SPF chickens

To explore the antibody responses and protective effects induced by subgroup J avian leukosis virus (ALV-J) gp85 protein vaccine plus different adjuvants (CpG and white oil adjuvant YF01) combined with the immune enhancer Taishan Pinus massoniana pollen polysaccharide (TPPPS), we immunized SPF chickens with the recombinant ALV-J gp85 protein, along with different adjuvants and immune enhancer, which protected the chickens by inducing different levels of protective antibodies. Results showed that a single injection of gp85 recombinant protein could only produce low-titre antibodies that were maintained over a short time in few chickens. When combined with YF01 or CpG adjuvants, the recombinant protein could induce high-titre antibodies in most of the immunized chickens. Moreover, when the immune enhancer TPPPS was used with the two adjuvants, it further elevated the antibody levels for a longer duration. The eggs from four groups with the highest levels of ALV-J antibodies were collected, hatched, and examined for maternal antibodies. The protection by the maternal antibodies against ALV-J infection in the TPPPS-immunized group was higher than that in the group without TPPPS, which was consistent with the observations in the parents. This study shows that the immune enhancer TPPPS, combined with YF01 or CpG adjuvants, can enhance the immunogenicity of gp85 recombinant proteins, and provide a better immuno-protection. It provides a powerful experimental basis for the development of ALV-J subunit vaccine. Efficient subunit vaccine development will also accelerate the process of purification of ALV-J.     

以重组融合蛋白为基础的钩端螺旋体酶联免疫吸附试验检测试剂盒的初步研制

目的 建立以钩端螺旋体融合蛋白LipL32-LipL41(LipL32:脂蛋白L32,lipoprotein32;LipL41:脂蛋白L41,lipoprotein41)为包被抗原的酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测试剂。方法 选择问号钩端螺旋体LipL32和LipL41作为研究靶标,采用连接引物PCR法构建lipL32-lipL41融合基因,原核表达获得重组蛋白,以该蛋白为包被抗原建立ELISA检测体系,对检测体系的灵敏度、特异性、稳定性等性能指标进行评价。结果 扩增获得了1 900 bp左右的lipL32-lipL41融合基因,原核表达出了90 kDa左右的重组融合蛋白LipL32-LipL41,建立了基于LipL32- LipL41的ELISA检测体系,采用77例确诊病例和85例阴性对照血清进行初步验证,检测体系的灵敏度为96.1%,特异度为97.6%,粗一致性为96.9%,约登指数为0.937。结论 以LipL32-LipL41重组融合蛋白为包被抗原建立ELISA免疫检测初步验证的灵敏度、特异性和稳定性较好,可进行下一步验证工作。