减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…     

EB病毒疫苗的研究进展

EB病毒是一种感染最广泛的人类γ疱疹病毒,与传染性单核细胞增多症、鼻咽癌、Hodgkin’s淋巴瘤以及Burkitt淋巴瘤等发生密切相关。预防和治疗EB病毒相关疾病一直是研究的热点,预防性疫苗主要以病毒包膜糖蛋白gp350为靶点,刺激机体产生抗体以阻止病毒感染;已有多种疫苗进入临床试验,用于预防传染性单核细胞增多症和器官移植后淋巴增殖性紊乱等疾病,并取得良好的试验结果。治疗性疫苗则以病毒复制感染过程中表达的病毒核抗原(EBNAs)和潜伏膜蛋白(LMP1和LMP2)为免疫治疗靶点,刺激机体产生特异性细胞免疫应答,增强细胞毒性T细胞杀伤肿瘤作用。国内外均已有疫苗进入针对鼻咽癌等肿瘤免疫治疗的临床试验,试验结果表明疫苗兼具良好的安全性和免疫原性。本文还综述EB病毒疫苗研究的最新方法、策略和成果,并探讨其中可借鉴的技术和思路,为EB病毒疫苗的研究提供潜在可行的新途径。     

森林脑炎病毒地鼠肾细胞培养增殖动态及疫苗生产工艺改进的研究

为了解森林脑炎病毒的增殖动态,改进现有疫苗的工艺,将森林脑炎病毒感染地鼠肾细胞,控制不同的培养条件,以利病毒增殖,并于不同时间收获病毒液,测定病毒滴度。疫苗液用不同浓度的福尔马林灭活后,测定疫苗的保护指数。结果表明森张株病毒增殖高峰在68－72h,多次收获的病毒液及其病毒滴度、疫苗效力均优于现有疫苗,新工艺可提高病毒产量,为疫苗的精制纯人奠定了基础。     

中国脊髓灰质炎病毒疫苗株基因特征及其神经毒力的初步观察

中国脊髓灰质炎病毒疫苗株普遍存在基因变异的现象,如重组,点突变等。我们选出１０株有代表性的变异株,在具有人脊灰病毒受体基因的转基因小鼠ＰＶＲ－Ｔｇ２１中做毒力分析,发现Ｓａｂｉｎ １基因型与野毒基因型的重组株显示很强的神经毒力,其ＰＤ５０ｉｎＴＣＩＤ５０值为４．５,而Ｓｂｉｎ１标准株的ＰＤ５０值大于８０。另外两株Ｉ型疫苗株只有关键性核苷酸位点发生突变,只在位点５２５－发生突变的一株,其ＰＤ５０值为     

HIV-1 gagpol重组腺病毒疫苗的构建及其免疫原性的研究

目的：构建HIV-1重组腺病毒疫苗并初步鉴定其免疫原性。方法：用Adeno-XExpressionSystem试剂盒将HIV—lgagpol基因片段插入到腺病毒载体上,通过脂质体介导将获得的重组腺病毒质粒Adeno—X-gagpol转染至293细胞,使之自主包装成有感染活性的重组腺病毒tad—gagpol,用western-blotting法鉴定其表达情况;用CsCl密度梯度离心法纯化该重组腺病毒,以5×10^8 pfu／mL的滴度lmL单次免疫小鼠,15天后测小鼠体液免疫效果。结果：克隆序列与设计相符,重组腺病毒疫苗能稳定表达gag—pol蛋白,体液免疫中检测出抗p55和p24的特异性抗体。结论：HIV—lgagpol重组腺病毒构建成功,具有一定的体液免疫原性。     

增强DNA疫苗免疫应答的新进展

DNA疫苗为编码抗原蛋白的真核表达载体,注入体内后在原位表达所编码的抗原并诱导免疫应答,在预防感染、治疗自身免疫性疾病、过敏性疾病和肿瘤等疫病中有着很好的应用前景。但与灭活疫苗相比,其免疫效价还比较低。有多种策略能够增强或调节DNA疫苗诱导的免疫应答,其中,作为外源基因载体的质粒的组成及插入的有关基因均可直接或间接地影响免疫反应的效果,在构建DNA疫苗质粒时,加入细胞因子、融合信号、泛素等基因以及ISS序列,另外还可以通过设计一些对抗原提成细胞有影响的分子共注射,以及加入转移分子,都可以明显增强DNA疫苗的免疫效果,从而有利于研制更有效的DNA疫苗。     

微载体灌注培养制备Vero细胞狂犬病疫苗

本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术。在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6~7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0~8.5logLD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到《中国生物制品规程》2000年版要求。实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行。     

A novel Helicobacter pylori cell-surface polysaccharide

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.     

Recombinant Sendai virus induces T cell immunity against respiratory syncytial virus that is protective in the absence of antibodies

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and a vaccine is highly desirable. The fusion (F) protein of RSV is an important vaccine target, but the contribution of F-specific T cells to successful vaccination remains unclear. We studied the immune response to vaccination of mice with a recombinant Sendai virus expressing RSV F (rSeV F). rSeV F induced protective neutralizing antibody and RSV F-specific CTL responses. T cell immunity was stronger than that induced by recombinant vaccinia virus (rVV F), a well characterized reference vector. Vaccination of antibody-deficient mice showed that vaccine-induced RSV F-specific T cells were sufficient for protective immunity. rSeV F induced T cell immunity in the presence of neutralizing antibodies, which did not impair the vaccine response. Although the F protein only contains a subdominant CTL epitope, vaccination with rSeV F is sufficient to induce protective T cell immunity against RSV in mice.     

Recombinant chimpanzee adenovirus vector vaccine expressing the spike protein provides effective and lasting protection against SARS-CoV-2 infection in mice

SARS-CoV-2 infection is a global public health threat. Vaccines are considered amongst the most important tools to control the SARS-CoV-2 pandemic. As expected, deaths from SARS-CoV-2 infection have dropped dramatically with widespread vaccination. However, there are concerns over the duration of vaccine-induced protection, as well as their effectiveness against emerging variants of concern. Here, we constructed a recombinant chimpanzee adenovirus vectored vaccine expressing the full-length spike of SARS-CoV-2 (AdC68-S). Rapid and high levels of humoral and cellular immune responses were observed after immunization of C57BL/6J mice with one or two doses of AdC68-S. Notably, neutralizing antibodies were observed up to at least six months after vaccination, without substantial decline. Single or double doses AdC68-S immunization resulted in lower viral loads in lungs of mice against SARS-CoV-2 challenge both in the short term (21 days) and long-term (6 months). Histopathological examination of AdC68-S immunized mice lungs showed mild histological abnormalities after SARS-CoV-2 infection. Taken together, this study demonstrates the efficacy and durability of the AdC68-S vaccine and constitutes a promising candidate for clinical evaluation.