Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

Evaluation of immune responses induced by SAG1 and MIC3 vaccine cocktails against Toxoplasma gondii

The aim of this study was to evaluate the immune responses of a SAG1 and MIC3 vaccine cocktail in BALB/c mice. Ninety-six BALB/c mice were randomly divided into eight groups, including three plasmid DNA vaccine groups (pcDNA-MIC3, pcDNA-SAG1, pcDNA-MIC3+pcDNA-SAG1), three recombinant pseudotype baculovirus vaccine groups (BV-G-MIC3, BV-G-SAG1, BV-G-SAG1+BV-G-MIC3) and two control groups (PBS and BV-G-EGFP). All groups were immunized intramuscularly twice at three-week intervals. The production of anti-Toxoplasma gondii lysate antigen (TLA) antibodies, lymphoproliferation, levels of IFN-γ, IL-4 and IL-10 and the survival time were monitored after vaccination. The results showed that immunization of BALB/c mice with MIC3 and SAG1 vaccines stimulated both the cellular and humoral immune responses with the production of anti-T. gondii TLA antibodies. The vaccine cocktails of pcDNA-MIC3+pcDNA-SAG1 or BV-G-SAG1+BV-G-MIC3 induced significantly higher immunogenicity than a single-gene vaccine (P<0.05). Splenocytes from the immunized mice significantly proliferated in response to the TLA and released interferon (IFN)-γ (P<0.05). However, the levels of IL-4 and IL-10 in the sera of the immunized mice were not significantly different from those of the controls (P>0.05). Immunization with the vaccine cocktail (BV-G-SAG1+BV-G-MIC3) in mice significantly prolonged survival (50%; P<0.05) against a lethal challenge of T. gondii (RH tachyzoites), while all mice in the other immunized groups and control groups died within 20 and 4 days post-infection, respectively. Furthermore, the recombinant pseudotype baculovirus vaccines induced better immunogenicity than the plasmid DNA vaccines (P<0.05). These results suggest that an excellent vector-mediated vaccine cocktail strategy might be used to develop a new generation of vaccines against T. gondii infection.     

新孢子虫与弓形虫交叉抗原AMA1基因重组腺病毒的构建及免疫应答分析

为构建新孢子虫和弓形虫AMA1基因重组腺病毒,并分析其免疫原性,本试验根据新孢子虫和弓形虫AMA1基因序列的开放阅读框,设计新孢子虫和弓形虫交叉抗原AMA1基因通用引物,构建重组克隆质粒pMD18T-NcAMA1、pMD18T-TgAMA1及重组腺病毒穿梭质粒ADV4-Nc/TgAMA1,将ADV4-Nc/TgAMA1和骨架质粒pacAd5线性化后共转染293T细胞,包装Ad5-Nc/TgAMA1重组腺病毒,测定病毒滴度后,收集病毒液接种BALB/c小鼠,间接ELISA检测小鼠血清IgG抗体水平。结果显示,Nc/TgAMA1在Ad5-Nc/TgAMA1重组腺病毒中获得表达,测定Ad5-Nc/TgAMA1重组腺病毒滴度为10⁹PFU/mL,接种BALB/c小鼠后,Ad5-Nc/TgAMA1接种组IgG抗体水平明显高于pVAX1-Nc/TgAMA1质粒组和PBS对照组。结果表明,构建的Ad5-Nc/TgAMA1重组腺病毒能诱导小鼠产生特异性体液免疫应答。本试验为新孢子虫和弓形虫交叉抗原AMA1基因重组腺病毒载体疫苗的研制奠定了基础。     

Construction and Analysis of Immune Response of Neospora caninum and Toxoplasma gondii Cross Recombinant Adenovirus Antigen AMA1 Gene

In order to establish AMA1 recombinant adenovirus of Neospora caninum (N.caninum) and Toxoplasma gondii (T.gondii), and analyze the immunogenicity of it, cross universal primers were designed according to the open reading frame of N. caninum and T. gondii AMA1 gene sequences. Based on pMD18T-NcAMA1 and pMD18T-TgAMA1 cloning plasmid, recombinant adenovirus shuttle plasmid ADV4-Nc/TgAMA1 was constructed. Then, ADV4-Nc/TgAMA1 and pacAd5 backbone plasmid were linearized and co-transfected 293T cells. After packaging recombinant adenovirus and measuring the virus titer, collected virus was inoculated into BALB/c mice, confirmed the IgG antibody levels by indirect ELISA method. The results showed that Nc/TgAMA1 was expressed in Ad5-Nc/TgAMA1 recombinant adenovirus, Ad5-Nc/TgAMA1 recombinant adenovirus titer was 10⁹ PFU/mL. IgG antibody levels in the Ad5-Nc/TgAMA1 vaccinated group were significantly higher than pVAX1-Nc/TgAMA1 plasmid group and PBS control group. This result indicated that the constructed Ad5-Nc/TgAMA1 recombinant adenovirus could induce specific humoral immune response in mice, this research laid a solid foundation for the development of a recombinant adenovirus vaccine against N. caninum and T. gondii.     

基因枪与肌肉注射猪繁殖与呼吸综合征病毒ORF5基因疫苗诱导小鼠体液及细胞免疫的比较研究

猪繁殖与呼吸综合征病毒ORF5基因疫苗（pcDNA—PRRSV—ORF5）以不同免疫途径（基因枪和肌肉注射）免疫BALB／c小鼠，以PBS和空载质粒pcDNA3．1（＋）为对照，采用流式细胞仪（FACS）、淋巴细胞增殖试验（MTT法）及间接ELISA试验分别对小鼠外周血中CD4^＋、CD8^＋T淋巴细胞数、T淋巴细胞的转化功能及小鼠血清中特异性PRRSV血清抗体IgG动态变化进行了检测。结果表明，pcDNA—PRRSV-ORF5基因疫苗接种小鼠后外周血对ConA有明显的反应性，试验组与对照组比较差异极显著（P〈0．01）或显著（P〈0．05），CD4^＋T淋巴细胞数在免疫后7d高于对照组（P〈0．01），CD8^＋T淋巴细胞在免疫后28d高于对照组，不同途径基因疫苗接种小鼠后均诱导小鼠产生PRRSV特异性IgG。在诱导细胞免疫方面，基因枪和肌肉注射各组间无明显差异；在诱导体液免疫方面，基因枪法优于肌肉注射。研究表明制备的pcDNA—PRRSV—ORF5基因疫苗免疫小鼠能够诱导其机体产生良好的体液和细胞免疫应答，基因枪法较肌肉注射更能诱导体液免疫应答的产生。     

pVAX1/SjTSP2 DNA疫苗免疫预防小鼠日本血吸虫病的效果评估

为评估SjTSP2分子作为日本血吸虫DNA疫苗候选抗原分子的潜力,本实验扩增了编码该分子的DNA片段,构建了重组真核质粒pVAX1/SjTSP2,检测其在哺乳动物细胞中表达情况后,通过基因枪轰击法免疫小鼠,应用ELISA、流式细胞术等检测重组质粒诱导的免疫反应,计算减虫减卵率,评估对小鼠日本血吸虫病的免疫保护效果。间接免疫荧光试验结果显示该质粒能在293T细胞中表达。质粒免疫小鼠诱导了显著升高的特异性IgG抗体和IFN-γ、IL-4等细胞因子。小鼠免疫保护试验结果表明,免疫组小鼠虫荷数较PBS对照组增加12.4%(P=0.368),但肝组织虫卵减少10.01%(P=0.486)。说明制备的DNA疫苗能刺激小鼠产生较强的免疫反应,但诱导的免疫保护作用并不理想,为血吸虫DNA疫苗研究提供了参考数据。     

表达猪附红细胞体ENO基因的质粒DNA的构建及免疫效果研究

试验旨在构建表达猪附红细胞体ENO基因的质粒DNA,并测定其免疫效果。将猪附红细胞体ENO基因克隆到PVAX1真核表达载体上,然后转染到Vero细胞中进行表达并测定其免疫效果。将18只BALB/c小鼠（雌雄各半）随机分为3组（PVAX1-ENO质粒DNA免疫组、PVAX1空载体对照组及PBS对照组）,每组6只,每组小鼠对应免疫接种PVAX1-ENO质粒DNA、PVAX1空质粒和PBS。分离血清并通过ELISA方法测定血清中ENO蛋白抗体效价、IgG₁和IgG_2a抗体水平及IFN-γ和IL-4细胞因子水平。三免2周后每组选取3只小鼠检测CD4⁺和CD8⁺含量。结果显示,本试验成功构建了PVAX1-ENO质粒DNA,经PCR和酶切鉴定正确,并能在Vero细胞中成功表达。PVAX1-ENO质粒DNA免疫组ENO蛋白抗体水平、IgG₁和IgG_2a抗体水平、IFN-γ和IL-4细胞因子水平及CD4⁺和CD8⁺含量均显著高于PVAX1空载体对照组及PBS对照组（P<0.05）。结果表明,PVAX1-ENO质粒DNA可显著提高BALB/c小鼠的体液免疫水平,并在一定程度上刺激BALB/c小鼠细胞免疫。     

热带无爪螨主要变应原Blo t 5和Blo t 21融合表达质粒诱导小鼠产生IgG和调节性T细胞

旨在开发防治热带螨过敏症的兽用核酸疫苗,本试验通过对热带无爪螨主要变应原Blo t 5和Blo t 21的基因序列进行密码子优化,构建了热带螨主要变应原融合基因的真核表达载体pVAX1-PADRE-Blo t 5-21-CpG。为验证该真核表达载体的免疫效果,采用皮下注射,以100 μg/100 μL量的pVAX1-PADRE-Blo t 5-21-CpG核酸免疫BALB/c小鼠(n=6),通过ELISA检测小鼠血清中特异性IgG、IgG1和IgG2a的水平变化;流式细胞术检测小鼠脾淋巴细胞中调节性T细胞的变化。结果显示,真核表达质粒pVAX1-PADRE-Blo t 5-21-CpG免疫小鼠后可诱导特异性IgG和IgG2a水平升高,且IgG2a/IgG1的比值升高,这暗示免疫后小鼠机体发生偏向Th1型的免疫反应。此外,该核酸疫苗可诱导小鼠产生较高水平的CD4⁺CD25⁺Foxp3⁺ T细胞,这表明该真核表达质粒免疫小鼠后可能会诱发免疫抑制。     

Immunogenicity and protective efficacy of two recombinant pseudorabies viruses expressing Toxoplasma gondii SAG1 and MIC3 proteins

Toxoplasma gondii is one of the most common parasitic pathogens in humans and warm-blooded animals, causing toxoplasmosis. One of the efficient ways to control this disease is immunization. In this study, two recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV-SAG1) and TgMIC3 (rPRV-MIC3) based on the PRV vaccine strain were developed by homologous recombination and used for immunizing BALB/c mice. Ninety BALB/c mice were randomly divided into five groups including four experimental groups (inoculated twice in 4 weeks interval with PRV TK-/gG-/EGFP+, rPRV-SAG1, rPRV-MIC3, rPRV-SAG1+rPRV-MIC3, respectively) and one control group (inoculated with medium). All mice vaccinated with rPRV developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-γ and IL-2 production. These results demonstrated that rPRV could induce significant humoral and cellular Th1 immune responses. Moreover, rPRV immunization induced partial protection against a lethal challenge with T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. The mice immunized with the rPRV-SAG1 and rPRV-MIC3 cocktail could develop higher T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge. These results suggested that expression of protective antigens of T. gondii in PRV is a novel approach towards the development of a vaccine against both animal pseudorabies and toxoplasmosis.     

丝状支原体山羊亚种LppA蛋白N末端基因真核表达载体构建及小鼠免疫效果分析

基于丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri,Mmc)贵州株的LppA蛋白N末端基因,构建真核重组表达质粒pVAX1-LppA,并对免疫效果进行分析,为防控羊支原体肺炎提供新思路.以构建的真核重组表达质粒pVAX1-LppA(50、100、150 μg)、pVAX1空载体、无菌...     

Vaccination with a novel multi-epitope ROP8 DNA vaccine against acute Toxoplasma gondii infection induces strong B and T cell responses in mice

Rhoptry proteins (ROPs) are involved in the cell invasion and parasitophorous vacuole (PV) formation and also vital for survival of Toxoplasma gondii (T. gondii) within host cells. ROP8 have a main role during the early phase of infection and can express in tachyzoite and bradyzoite forms. In the present study, we designed a novel multi-epitope DNA vaccine encoding the potential B and T-cell epitopes from ROP8 protein to evaluate the immunity and protective efficacy against acute T. gondii infection in BALB/c mice. For this purpose, several bioinformatics online servers were used. At first, the potential epitopes were selected for T and B cells using immune epitope database (IEDB) and BCPREDS online services. Then, the selected epitopes were fused together by SAPGTP linker. Finally, the physico-chemical features, secondary and tertiary structures, antigenicity, and allergenicity of the peptide were evaluated through different bioinformatics tools. Lastly, the multi-epitope peptide was successfully cloned into pcDNA3.1 expression vector. The DNA vaccine was subcutaneously injected into BALB/c mice and the immune responses of the vaccinated mice and controls were determined. The obtained results revealed that the multi-epitope ROP8 peptide has 183 amino acid residues with average molecular weight (MW) of 18.974 kDa. More than 98 % residues of the peptide were incorporated in favored and allowed regions of the Ramachandran plot. The antigenicity of multi-epitope peptide were estimated 0.8751 and 0.7649 by ANTIGENpro and VaxiJen servers, respectively. BALB/c mice immunized with DNA vaccine showed significantly increased the level of specific anti-T. gondii antibodies (P < 0.05), and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a TH1-type cellular immune response with production of IFN-γ and prolonged survival time, compared with the control groups (P < 0.05). The findings revealed that the multi-epitope ROP8 DNA vaccine induced strong humoral and cellular responses and prolonged the survival time in BALB/c mice, suggesting selection of potential epitopes may be a promising strategy for the design of multi-epitope-based vaccines.     

携带新城疫病毒F基因DNA疫苗的减毒鼠伤寒沙门氏菌的构建及其免疫原性

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计1对引物,通过RT-PCR扩增出鹅源NDV分离株JS5F基因(约1700bp),测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1-F。pVAX1-F经脂质体转染COS-7细胞,间接免疫荧光试验检测出F基因在COS-7细胞中的表达产物。将pVAX1-F转化减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pVAX1-F)。重组菌以109CFU/只的剂量2次免疫BALB/c小鼠,免疫小鼠可以检测到特异性针对NDVF蛋白的血清抗体和小肠粘膜抗体应答,SL7207(pVAX1-F)免疫组抗体水平显著高于SL7207(pVAX1)组(P<0.05)。将SL7207(pVAX1-F)以109CFU/只剂量口服免疫1日龄雏鸡,免疫保护试验结果显示,SL7207(pVAX1-F)免疫组对鸡具有良好的保护率(77.27%),与空白对照组和SL7207(pVAX1)空载体组之间存在显著性差异(P<0.05)。结果表明,该运送DNA疫苗的减毒沙门氏菌系统在体内能成功释放所携带的质粒,并能刺激机体产生免疫应答,可对NDV强毒攻击提供良好的免疫保护作用,提示该疫苗候选株对新城疫的控制有重要应用前景。     

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T_h1 and T_h2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

A recombinant DNA vaccine encoding C. andersoni oocyst wall protein induces immunity against experimental C. parvum infection

Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a cause of reduction of milk production in dairy cow. In this study, a novel chimeric DNA vaccine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum was determined. BALB/c mice were divided into 3 groups and immunized with DNA vaccine expressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid containing the AB gene, respectively. After inoculation of 1 × 10(6) oocysts of C. parvum, the humoral and cellular immune responses were detected. Experimental results showed that the recombinant plasmid can induce corresponding specific antibody response, simultaneously influenced cellular immune responses, and provided greater protection rate (48.6%) than the other groups. These results indicated that chimeric DNA vaccine has a potential in Cryptosporidium vaccine development.     

奶牛乳腺炎金黄色葡萄球菌ClfA核酸疫苗的构建及免疫原性试验

扩增了奶牛乳腺炎金黄色葡萄球菌ClfA基因,并将其克隆至真核表达载体pVAX1启动子下游,构建成真核表达质粒,通过体外细胞转染试验,运用IFA方法进行抗原性初步确认,所构建的重组DNA疫苗质粒能在真核细胞中表达并被金黄色葡萄球菌抗体特异性识别。为进一步评价侯选疫苗的免疫原性,进行了BALB/c小鼠免疫试验,分别检测免疫后的ELISA抗体水平、Th1/Th2类细胞因子水平以及T淋巴细胞增殖试验。结果表明,构建的核酸疫苗pVAX1-ClfA免疫小鼠后,ELISA抗体水平提高,Th1/Th2类细胞因子含量提升,T细胞增殖能力增强。     

IL-18增强日本血吸虫DNA疫苗Sj23的免疫保护效果

将小鼠IL-18基因和日本血吸虫Sj23膜蛋白基因分别插入pVAX1载体,构建真核表达质粒pVAX/mIL-18和pVAX/Sj23,联合或单独免疫小鼠,以pVAX1空载体作对照,免疫2次,间隔2周,2次免疫后4周攻击日本血吸虫尾蚴.体液和细胞免疫检测结果表明,联合免疫组能诱导小鼠产生较强的抗日本血吸虫成虫可溶性抗原(SWAP)IgG,较高水平的IFN-γ和IL-2.攻虫试验表明,联合免疫小鼠成虫减虫率和肝脏减卵率分别达41.6%和49.4%,明显高于pVAX/Sj23单独免疫组(减虫率和肝脏减卵率分别为26.5%和41.4%).以上试验结果表明,IL-18能明显增强Sj23 DNA疫苗在小鼠体内的免疫应答,并且产生较强的保护作用.     

Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p < 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge.     

牛新孢子虫NcSAG1基因工程亚单位疫苗的免疫试验

为了解新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗对实验动物的免疫效果,本试验提取延边黄牛新孢子虫野毒株基因组DNA,用PCR技术扩增新孢子虫NcSAG1表面蛋白基因,并在大肠杆菌中进行原核表达,将Western-blot鉴定具有免疫活性的重组蛋白与弗氏佐剂混合制备NcSAG1基因工程亚单位疫苗,免疫BALB/c小鼠,应用间接ELISA方法测定体液免疫水平,应用流式细胞技术测定细胞免疫水平,以此评价疫苗对实验动物的免疫应答反应。结果显示,制备的NcSAG1基因工程亚单位疫苗免疫BALB/c小鼠后,在三免后第3天时,检测抗体的OD450nm值达0.688;CD4+/CD8+值达3.650,均显著高于重组蛋白免疫组和PBS对照组,说明制备的基因工程亚单位疫苗能够提高实验动物的体液免疫和细胞免疫水平。本试验为牛新孢子虫NcSAG1表面蛋白基因工程亚单位疫苗的深入研究奠定了基础。     

密码子优化增强新城疫病毒DNA疫苗的表达水平和免疫效果

为提高新城疫病毒（Newcasti Disease Virus,NDV）F基因DNA疫苗的表达量,增强其免疫效果。按照鸡体内偏嗜性密码子人工合成了NDV F48E9株的F基因optiF,插入到真核表达载体pVAX1中获得pVAX1-optiF。将F48E9株的F基因pVAX1-F与pVAX1-optiF分别转染COS-7细胞,72h后间接免疫荧光和Western blot检测细胞中瞬时表达的F蛋白。将质粒pVAX1、pVAX1-F和pVAX1-optiF以200μg／只的剂量分别股四头肌多点注射18只2周龄SPF鸡,2周后加强免疫1次,同时设立PBS对照。二免2周后每组取12只鸡以100EID50的F48E9株NDV强毒进行攻毒,评价这2种DNA疫苗的免疫效果。结果表明,F基因密码子的优化可显著提高NDVDNA疫苗诱导的保护性体液免疫和细胞免疫应答水平,攻毒后所有pVAX1-optiF免疫鸡均获得保护（12／12）,而pVAX1-F组保护率只有17％（2／12）。DNA疫苗的免疫效果与抗原基因的表达量及表达抗原的免疫原性有直接关系。与未经修饰的F基因相比,修饰后的F基因体外瞬时表达水平明显提高。