表达猪圆环病毒II型ORF2基因的重组猪伪狂犬病病毒的免疫原性

摘要:【目的】为研制预防猪圆环病毒II型(PCV2)感染的重组伪狂犬病病毒(PRV)活载体疫苗。【方法】将PCV2 ORF2基因插入到PRV通用载体pG中,利用脂质体LipofectamineTM 2000试剂盒将重组转移质粒pGO与猪PRV弱毒HB98株DNA共转染猪睾丸(ST)细胞,通过3轮蚀斑纯化重组病毒。将重组病毒、商品化PCV2灭活苗及DMEM培养液分别免疫6周龄雌性昆明小鼠,4周后加强免疫1次,首免后第8周用PCV2强毒NY株对小鼠进行攻毒。【结果】成功获得表达ORF2基因的重组病毒PGO,首免重组病毒后小 鼠体内抗PCV2的ELISA抗体水平很低,二免后小鼠PCV2特异的ELISA抗体水平明显升高,并且重组病毒组能够激发PCV2特异的淋巴细胞增殖效应。攻毒试验表明重组病毒组和PCV2灭活疫苗组均能有效抵抗PCV2强毒攻击。【结论】表明表达ORF2基因的重组病毒PGO具有良好免疫原性。     

表达鸡传染性法氏囊病毒VP2蛋白的干酪乳杆菌免疫保护效力

利用干酪乳杆菌作为传染性法氏囊病毒(IBDV)VP2抗原传递系统,探讨口服雏鸡的免疫次数、免疫剂量、免疫途径和攻毒保护效果。用pLA-VP2重组干酪乳杆菌对5日龄雏鸡进行二次和三次免疫,并设108、109、1010 CFU/mL的重组干酪乳杆菌组,间接ELISA检测血清IgG和小肠洗液sIgA,末免后7 d攻毒,计算保护效果。根据确定的2次免疫和109 CFU/mL免疫剂量免疫5日龄雏鸡,分别口服、滴鼻/点眼pLA-VP2/L.casei,口服、肌注商品活苗及口服pLA/L.casei和PBS为对照,监测IgG和sIgA抗体水平;末免后7 d检测脾淋巴细胞增殖情况并攻毒,7 d后剖检,观察法氏囊损伤程度并记录病变得分和保护率。结果表明各组的特异性sIgA、IgG抗体水平显著高于对照组(P0.01);口服pLA-VP2/L.casei组的淋巴细胞刺激指数显著高于其他组(P0.01),保护率高达83.3%,免疫保护效果优于滴鼻/点眼组。因此,构建的重组干酪乳杆菌的安全性优于商品活苗,可以作为IBDV候选疫苗。     

表达PEDV S和PoRV VP7蛋白的重组伪狂犬毒株构建

【背景】猪流行性腹泻、猪轮状病毒病与猪伪狂犬病是严重危害全球养猪业的3种重要传染病,混合感染往往导致猪场更严重的损失。【目的】利用同源重组技术构建共表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV) S蛋白和猪轮状病毒(Rotavirus,PoRV) VP7蛋白的猪伪狂犬三联基因工程疫苗株,并研究其部分生物学特性。【方法】通过序列比对、蛋白结构分析筛选s基因的475?804 aa和vp7基因的17?339 aa作为毒株构建的目的片段,依次构建了pMD-S、pMD-VP7、pMD-VP7.S克隆载体和pEGFP-VP7.S转移载体。将质粒pEGFP-VP7.S和PRV XJ亲本株同源重组,空斑纯化得到重组毒株PRV (CM),对其稳定性和增殖特性进行研究。【结果】构建了共表达S蛋白和VP7蛋白的伪狂犬基因工程病毒,连续传代20次,均能检测到vp7和s基因,而gE基因阴性;Western blotting证实2种外源基因在重组病毒中均能实现良好的表达;测定亲本毒株和重组毒株的TCID50分别是10?7.59/0.1 mL和10?7.25/0.1 mL。【结论】获得了伪狂犬基因工程重组弱毒株PRV (CM),外源基因稳定存在,毒力基因稳定缺失,增殖特性差异不大,为PRV、PEDV和PoRV基因工程三联苗研究奠定了基础。     

共表达PCV2 ORF2和猪IL-18基因的重组猪伪狂犬病毒对小鼠的免疫原性

【目的】研制猪伪狂犬病毒(PRV)和猪圆环病毒2型(PCV2)的二联活疫苗,并用猪IL-18作为免疫佐剂。【方法】将猪IL-18基因插入到质粒p GO中,获得的重组转移质粒p GO18与猪PRV弱毒HB98株DNA共转染ST细胞,并进行空斑筛选和纯化;RT-PCR和Western blot分别从转录和蛋白水平鉴定其表达情况。将重组病毒PGO18和PGO、PRV弱毒株HB98、PCV2灭活商品苗和1640细胞培养基分别免疫6周龄雌性昆明小鼠,4周后二次免疫,二免后4周用PCV2 DF强毒和PRV Min/A强毒接种小鼠。通过ELISA、血清中和试验和流式细胞术及攻毒保护试验评价重组病毒的免疫原性。【结果】获得了重组病毒PGO18,并且可在ST细胞内表达;PGO18可诱导小鼠机体产生PCV2的ELISA和PRV的中和抗体水平,刺激CD3+、CD4+、CD8+T细胞亚群的增殖,且能有效抵抗PCV2和PRV强毒攻击。【结论】IL-18基因可增强重组病毒的免疫效果,使重组病毒具有良好的免疫原性,有望成为防治PCV2和PRV的候选疫苗株。     

A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate

Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK⁻/gE⁻/LacZ⁺ mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK⁻/gE⁻/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

Eimeria tenella: construction of a recombinant fowlpox virus expressing rhomboid gene and its protective efficacy against homologous infection

A recombinant fowlpox virus (rFPV) expressing the Eimeria tenella rhomboid gene was constructed and its protective efficacy against homologous infection in chickens determined. Three-day-old-specific pathogen free (SPF) chickens were immunized s.c. with 10(2) plaque forming units (PFU), 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, and challenged with 5x10(4) homologous sporulated oocysts 14 days post-immunization (p.i.). The specific antibody response and lymphocyte proliferation were measured 1, 2, 3 and 4 weeks p.i. Oocyst output, body weight gains and lesion scores were measured to evaluate the protective effects of immunization. rFPV-rhomboid elicited a specific humoral immune response and stimulated proliferation of peripheral blood lymphocytes. The lesion scores in groups vaccinated with rFPV-rhomboid were significantly higher than in other groups. At the same time, rFPV-rhomboid improved body weight significantly compared with other groups. Immunization with rFPV-rhomboid reduced oocyst shedding significantly, resulting in a protection rate of 39.6%, 41.1% or 41.7% given a dose of 10(2) PFU, 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, respectively. These results indicated that rFPV can induce immune responses and offer partial protection of chickens against E. tenella challenge.     

Immunogenicity of DNA and recombinant Sendai virus vaccines expressing the HIV-1 gag gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens. Foundation item: National 863 project (2003AA219070)     

表达O型口蹄疫病毒VPl基因的重组病毒BHV-1的构建与鉴定

[目的]为了构建表达口蹄疫病毒(O/china/99)VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒(CMV)启动子之下构建gE基因缺失转移载体.[方法]利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒.通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1.[结果]PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Westem blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达.[结论]本研究成功地构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础.     

表达O型口蹄疫病毒 VP1基因的重组病毒BHV-1的构建与鉴定

摘要：【目的】为了构建表达口蹄疫病毒（O/China/99）VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒（CMV）启动子之下构建gE基因缺失转移载体。【方法】利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1。【结果】PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。【结论】本研究成功的构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).     

A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice

The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 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-gamma and IL-2 production. And the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.     

鹅细小病毒和番鸭细小病毒核酸疫苗重组质粒的构建及表达

将鹅细小病毒（GPV）和番鸭细小病毒（MPV）主要结构蛋白（VP2－VP3）基因克隆到核酸疫苗质粒pIRESlneo载体上,构建了核酸疫苗重组质粒pIGVP1和pIMVP,通过脂质体转染法分别将重组质粒到鹅胚成纤维细胞和番鸭胚成纤维细胞中,核酸疫苗重组质粒pIGVP1和pIMVP分别转染鹅胚成纤维细胞和番鸭成纤维细胞中,于转染后72h收取细胞,细胞裂解液裂解后,经Western blot检测其表达产物可出现特异性反应带,证明表达产物具有很好的反应原性。     

Immunogenicity of a recombinant MVA and a DNA vaccine for Japanese encephalitis virus in swine

We previously reported that mice immunized with recombinant modified vaccinia virus Ankara (MVA) encoding Japanese encephalitis virus (JEV) prM and E genes were completely protected against JEV challenge (Nam, J.H., Wyatt, L.S., Chae, S.L., Cho, H.W., Park, Y.K., Moss, B. Vaccine 1999,17: 261-268). In this study, we examined the immunogenicity in swine of this recombinant MVA (vJH9) or a DNA vaccine (pcJH-1) expressing the same JEV genes. Although the booster effect in mice with a combination of vJH9, pcJH-1 and inactivated JEV commercial vaccine was not apparent by measuring JEV antibodies, the recombinant MVA vaccine (vJH9) and the DNA vaccine (pcJH-l) efficiently produced neutralizing antibodies in swine and 2 doses of each showed a booster effect in mice and swine. Therefore, both vJH9 and pcJH-1 are good candidates for a second generation JEV vaccine.     

Immunogenicity of the capsid protein VP2 from porcine parvovirus expressed in low alkaloid transgenic tobacco

Porcine parvovirus is a widespread infectious viral disease with serious consequences to the reproductive health of swine. We have expressed the VP2 capsid protein of porcine parvovirus in the leaves of low alkaloid transgenic tobacco at approximately 0.3% of total soluble protein. Self-assembled virus-like particles were observed in planta by electron microscopy. Total soluble protein was extracted from the plant tissue and administered to mice by subcutaneous injection. An immune response was detected in these mice. The ability of serum antibodies to neutralize the infectivity of porcine parvovirus was further examined by a serum neutralization assay and was determined to be 1:2700–1:3900, a clear indication of the potential of VP2 expressed in plant material as a subunit vaccine against porcine parvovirus.     

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.     

Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII-β-galactosidase fusion protein

An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli β-galactosidase (ßGal). Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E. coli lacZ gene in a bacterial expression vector. The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA. The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein. One such mutant virus, PRV-Z1, was chosen for further analysis. PRV-Z1 expressed a glycosylated gIII-ßGal fusion protein after infection of PK15 cells. The fusion protein has no demonstrable ßGal activity and, although glycosylated, remains sensitive to the enzyme endo-β-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed.     

表达H3N2亚型猪流感病毒血凝素蛋白的重组腺病毒在猪体内的免疫试验

摘要:【目的】本研究旨在评价表达H3N2亚型猪流感病毒血凝素蛋白的重组腺病毒在猪体内的免疫效果。【方法】以10-8 TCID50、2×10-8 TCID50和4×10-8 TCID50的剂量经肌肉注射接种后2到6周每周检测血凝抑制（HI）抗体;比较肌肉注射、滴鼻、灌胃三种接种途径对仔猪免疫效果的影响,并通过攻毒保护试验进一步考察重组腺病毒rAd-HA-GFP在猪体内的免疫效力。【结果】以不同的剂量分别经肌肉注射免疫后, HI抗体水平与接种剂量呈正相关。三种接种途径均能刺激仔猪产生特异性抗体,肌肉注射组的HI抗体要高于其他两组,差异显著（P<0.01）。通过滴鼻和肌注注射方式进行攻毒后,阴性对照组仔猪在攻毒2d后出现体温升高、打喷嚏、咳嗽、流鼻液、精神沉郁、体重减轻等症状;而各免疫组仔猪未观察到明显的临床症状,各免疫组的病毒分离率也明显低于阴性对照组。【结论】重组腺病毒rAd-HA-GFP诱导的HI抗体达到1:320可以有效抵抗H3N2亚型猪流感病毒的侵袭,可以作为候选疫苗株。