为了选择合理的非洲猪瘟病毒(ASFV)重组载体的激发/加强免疫策略,构建表达ASFVCD2v-p30-p54融合抗原的重组伪狂犬病毒rPRV-F1,将其与表达相同融合抗原的重组腺病毒rAd-F1分为rAd-F1/rAd-F1、rAd-F1/rPRV-F1、rPRV-F1/rPRV-F1、rPRV-F1/rAd-F1 4 个组合进行猪免疫试验,分别于免疫后不同时间采血并分离血清进行ELISA抗体检测,分离的外周血单个核细胞(PBMC)经ASFV抗原刺激后进行IFN-y检测.免疫荧光与免疫转印试验结果显示,构建的rPRV-F1能在感染细胞中正确表达F1融合蛋白.所有免疫猪均能检测到抗原特异性抗体,加强免疫后的抗体水平显著升高.在测试的4个免疫方案中,尽管rAd-F1/rAd-F1组合初次免疫后的抗原特异性抗体水平和IFN-γ表达水平最高,但加强免疫后的抗体水平和IFN-y表达水平最低.与此相反,尽管rPRV-F1/rAd-F1组合初次免疫后的抗体水平和IFN-y表达水平最低,但加强免疫后的抗体水平和IFN-γ表达水平最高.这些研究结果表明rPRV/rAd是较好的ASFV重组载体疫苗的激发/加强免疫策略.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen affecting global swine industry. Our recent study has shown that the first four Ig-like domains of sialoadhesin (Sn4D) and the scavenger receptor cysteine-rich domains 5?9 (SRCR59) of CD163 can act as the soluble viral receptors (SVRs) of PRRSV. Co-injection with the two SVR-expressing recombinant adenovirus (rAd) vectors can protect pigs from the lethal challenge with three PRRSV strains. However, the in vivo expression of the two SVRs persists for only two weeks and thus their long-term anti-PRRSV effects remain to be improved. In this study, we fused the two SVRs with a flexible linker or self-cleaving peptide and expressed them with a single recombinant adenoassociated virus (rAAV) vector. The two rAAVs, namely rAAV-Sn4D-SRCR59-Fc and rAAV-SRCR59-Fc/Sn4D-Fc, were generated by using baculovirus-insect cell system. Western blotting analysis showed that the two SVR fusions were efficiently expressed in and secreted from the rAAV-transduced cells. Viral infection blocking assay showed that PRRSV titers in porcine alveolar macrophage (PAM) cells were reduced by 1.6?2.7 log10 after cocultivation with rAAV-Sn4D-SRCR59-Fc-transduced cells or by 1.9?3.2 log10 after co-cultivation with rAAVSRCR59-Fc/Sn4D-Fc-transduced cells. After single-dose injection of mice with the rAAV vectors, the expression of two SVR fusions persisted for at least 35 days, which was significantly longer than SRCR59-Fc expression in rAd-SRCR59-Fc-injected mice. Among the two SVR fusions expressed, both expression level and anti-PRRSV activity of SRCR59-Fc/Sn4D-Fc were higher than that of Sn4D-SRCR59-Fc. Therefore, rAAV-SRCR59-Fc/Sn4DFc generated can be developed as a novel anti-PRRSV reagent.

ObjectiveTo establish a receptor-binding capture method for recombinant adeno-associated virus (rAAV) purification. MethodsWe expressed polycystic kidney disease (PKD) domains 1 and 2 of AAV receptor in E. coli as an elastin-like polypeptide (ELP) fusion protein. We purified the fusion protein by inverse transition cycling (ITC). We generated two versions of rAAV-GFP and incubated them with ELP-PKD protein. We recovered the protein-bound rAAV-GFP by ITC and extracted the viral DNA for PCR analysis. We optimized the conditions for rAAV-GFP purification and identified the purified rAAV by electron microscopy and Western blotting. ResultsELP-PKD fusion protein was correctly expressed as a soluble protein which was purified to more than 90% purity. We demonstrated the specific affinity of ELP-PKD fusion protein for rAAV-GFP binding. We purified rAAV-GFP with 58% recovery from insect cells or 56% recovery from AAV-293 cells. After elution, we obtained final rAAV-GFP recovery rates of 46% and 44% from the two cell types, respectively. We demonstrated that the purified rAAV-GFP had the typical morphology and structural proteins of AAV. ConclusionWe established ELP-PKD-binding capture method for quick purification of rAAV from different cell types.

Abstract Toll-like receptor (TLR) ligands have emerged as the attractive adjuvant for subunit vaccines. However, selection of TLR ligands needs to be rationally chosen on the basis of antigen and adjuvant properties. In the present study, we expressed the Ag473 lipoprotein from Neisseria meningitides, flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) in Escherichia coli as single proteins and fusion proteins with VP2 protein of infectious bursal disease virus (IBDV). Both cellular and humoral adjuvanticities of the three TLR ligands were compared by immunization of mice in two different ways. Among the three co-administered TLR ligands, recombinant Ag473 lipoprotein exhibited the highest cellular and humoral adjuvanticities, including promotion of IL-4, IL-12, IFN-γ and IBDV VP2-specific antibody production. Among the three genetically fused TLR ligands, fusion with Ag473 D1 domain exhibited the highest cellular and humoral adjuvanticities. Overall, the adjuvanticities of genetically fused TRL ligands were significantly higher than that of co-administered TLR ligands. Fusion with Ag473 D1 domain exhibited superior adjuvanticity among the three TLR ligands delivered in two different ways.

为比较3种TLR配体对猪圆环病毒2型(PCV2)病毒样颗粒(VLP)疫苗的佐剂活性,本研究利用重组大肠杆菌表达脑膜炎奈瑟菌Ag473脂蛋白、创伤弧菌FlaB鞭毛蛋白和结核分枝杆菌热应激蛋白70(Hsp70),采用镍亲和柱纯化获得该3种重组蛋白;分别将相同剂量(10μg)3种重组蛋白、ISA206佐剂(100μL)和不完全弗氏佐剂(FIA)(100μL)与PCV2 VLP疫苗(50μg)混合,肌肉注射免疫小鼠,初免后14 d加强免疫1次;初免后每周采血分离血清,采用ELISA检测抗原特异抗体;加强免疫后14 d利用PCV2攻毒,攻毒后7 d和14 d采血分离血清,采用荧光定量PCR检测病毒DNA拷贝数;攻毒后14 d迫杀小鼠,制备并培养其脾细胞,经PCV2 VLP疫苗刺激后,采用试剂盒检测细胞因子的表达.SDS-PAGE分析结果显示,3种TLR配体在重组大肠杆菌中均获得正确表达,纯化重组蛋白的纯度大于90％.从初免14 d起,3个分子佐剂组中Ag473佐剂组的PCV2抗体水平最高,但低于ISA206佐剂组却高于FIA组,差异不显著(p>0.05).在PCV2攻毒前,3个分子佐剂组中Ag473佐剂组的小鼠脾细胞TNF-α和IFN-γ表达水平最高,但低于ISA206佐剂组却高于FIA组,差异显著(p<0.05);Hsp70佐剂组的IL-4表达水平最高,Ag473佐剂组次之,但均高于ISA206和FIA组.在攻毒后第7 d,5个免疫组小鼠的病毒拷贝数均较对照组显著下降(p<0.05),但免疫组间差异不显著;在攻毒后第14 d,5个免疫组小鼠的病毒拷贝数进一步下降,免疫组间差异不显著.研究结果表明在测试的3种分子佐剂中,Ag473脂蛋白具有较强的总体免疫佐剂活性,有望进一步开发为PCV2等病毒的亚单位疫苗佐剂.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen and vaccination is the primary tool for the disease control. Previously, we developed a more cost-effective PCV2 virus-like particle (VLP) vaccine by using ELPylation technology. In the present study, we compared the ELPylated VLP (ELP-VLP) PCV2 vaccine efficacy with commercial inactivated Yuanlijia vaccine and VLP-based Circoflex vaccine by experimental challenge. After one dose of vaccination with the three different vaccines, ELP-VLP vaccine group showed significantly (p < 0.05) stronger virus neutralizing antibody and interferon-γ responses than the two commercial vaccine groups. All vaccinated pigs showed significant (p < 0.05) improvement in average daily weight gain (ADWG) before challenge. After challenge with PCV2, however, only ELP-VLP-vaccinated pigs showed significant (p < 0.05) improvement in ADWG. All vaccinated pigs showed significant (p < 0.05) reductions in PCV2 loads in the blood, nasal secretion and lymph nodes, ELP-VLP-vaccinated pigs in particular. In addition, vaccination with ELP-VLP vaccine provided stronger protection against pulmonary and lymphoid pathologies than that with the two commercial vaccines. Therefore, ELP-VLP vaccine is more effective to control PCV2 infection than the two commercial vaccines based on clinical, immunological, virological and pathological evaluations.

Background Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). Methods The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. Results ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly ( p < 0.01) stronger VN antibody response and slightly ( p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. Conclusion A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.

Abstract Background Acute kidney injury (AKI) is one of the most common canine diseases with a high mortality rate. Neutrophil gelatinase-associated lipocalin (NGAL) is the novel biomarker for early diagnosis of renal injury. Only few sandwich ELISA kits are commercially available, all of which require the use of expensive enzyme-conjugated secondary or biotinylated antibody. The aims of this study were to develop high affinity monoclonal antibodies (mAbs) and simplified sandwich ELISA for canine NGAL detection. Results Recombinant canine NGAL was expressed in E. coli and purified to a high purity. Six hybridoma cell lines were generated by immunization of mice with the purified protein, all of which secreted high titers of specific mAbs. By screening 36 different antibody combinations, a pair of mAbs with high additivity and P/N ratio was selected as the capture and detection antibodies. By conjugation of the detection mAb with horse radish peroxidase, a simplified sandwich ELISA was developed with a correlation coefficient of 0.9939, detection limit of 8.28 ng/mL. The parallel test of 42 samples from healthy and AKI dogs showed the good agreement in NGAL concentrations detected by the simplified sandwich ELISA and commercial ELISA kit. Conclusions We developed canine NGAL-specific monoclonal antibodies and simplified sandwich ELISA. The simplified sandwich ELISA could replace the commercial ELISA kits for canine NGAL detection with the advantages of reduced cost and detection time.

Background: The pathological mechanism of ischemia/reperfusion acute kidney injury (I/R-AKI) differs from other forms of AKI. Neutrophil gelatinase-associated lipocalin (NGAL) is a sensitive biomarker for early diagnosis of AKI, but its utility for diagnosis of canine I/R-AKI remains to be evaluated. The aims of this study were to establish an I/R-AKI model in dogs and to evaluate the diagnostic value of NGAL for canine I/R-AKI. Methods: We randomly divided 12 beagle dogs into a sham and an I/R group. Artery and vein of the left kidneys of I/R group were cross-clamped for 60 min followed by reperfusion. The kidney samples were analyzed for histopathological lesions. Serum and urinary samples were analyzed for blood urea nitrogen (BUN), serum creatinine (sCr), serum NGAL (sNGAL), urinary creatinine (uCr), and urinary NGAL (uNGAL) analyses. Their detection sensitivities and specificities were compared using a receiver operating characteristics (ROC) method. The expression of NGAL in the renal tissues was analyzed by quantitative RT-PCR (qRT-PCR) and immunohistochemical (IHC) analyses. Results: After I/R, histopathological analysis showed typical AKI lesions in the dog kidneys of the I/R group, but not in the sham group. Compared to that of the sham group, BUN and sCr of the I/R group rose to significantly high levels from 24 h after I/R. Both uNGAL and sNGAL rose rapidly from 2 h, reached to the peak levels at 12 h, and then receded to the pre-operation levels by 72 h after I/R. The uNGAL/uCr ratio (uNCR) rose rapidly from 2 h and stayed at variably high levels from 6 to 60 h after I/R. The ROC analysis showed that detection sensitivities of uNCR, uNGAL, and sNGAL were significantly (P<0.0001) higher than that of sCr, without significant differences in specificity. The cut-off value of sNGAL, uNGAL, and uNCR were 14,642 pg/mL, 6,773 pg/mL, and 6,701 pg/mg, respectively. Both qRT-PCR and IHC analyses confirmed the dynamic expression of NGAL in the dog kidneys with ischemic acute kidney injury (I-AKI). Conclusions: There is potential for NGAL to be used as a sensitive biomarker for early prediction of canine I-AKI.

旨在获得非洲猪瘟病毒(ASFV)强免疫原性重组CD2v抗原,利用生物信息学软件进行CD2v抗原指数分析,将其细胞质内免疫显性区与类弹性蛋白多肽(ELP)在重组大肠杆菌中进行融合表达,对ELP-CD2v融合蛋白的相变循环(ITC)条件进行优化,在优化条件下进行融合蛋白纯化,利用烟草蚀纹病毒(TEV)蛋白酶活性包涵体切除ELP标签,通过免疫转印法对重组CD2v抗原进行鉴定,利用重组CD2v抗原建立ELISA抗体检测方法,与多抗原ELISA对ASFV抗体阳性和阴性血清进行平行检测.结果 显示,ELP-CD2v融合蛋白获得正确、可溶性表达,ITC条件为28℃和1.5 mol·L-1 NaCl,在0.2％ Triton X-100存在下进行ITC,纯化的融合蛋白纯度为76.3％;TEV蛋白酶活性包涵体能有效切割ELP标签,再次ITC回收的重组CD2v抗原纯度为91.7％,能被ASFV抗体识别;根据多抗原ELISA检测结果选择血清样品,用重组CD2v抗原ELISA进行检测,结果显示,15份ASFV抗体阴性血清均为CD2v抗体检测阴性,15份ASFV抗体阳性血清均为CD2v抗体检测阳性.这些研究结果表明,ASFV的CD2v蛋白胞内区存在强免疫原性表位,其重组抗原有望用于CD2v的抗体检测.

为建立敏感、特异的非洲猪瘟病毒(ASFV)抗体检测方法,将ASFV K205R基因与类弹性蛋白多肽(ELP)进行融合表达,用相变循环纯化ELP-K205R融合蛋白,用烟草蚀纹病毒(TEV)蛋白酶活性包涵体切除ELP标签,用相变循环回收K205R蛋白,用抗体阳性血清对重组K205R蛋白进行ELISA鉴定.结果 表明:重组大肠埃希菌能正确表达ELP-K205R融合蛋白,相变循环纯化的融合蛋白纯度大于80％;TEV蛋白酶活性包涵体能有效切割ELP标签,回收的重组K205R蛋白纯度大于95％,能被特异抗体阳性血清识别;重组K205R抗原与抗体阴性猪血清反应阴性,与抗体阳性猪血清反应阳性,D450nm值与血清稀释倍数具有线性相关性.用重组K205R抗原对已知猪血清进行ELISA检测,结果显示24份ASFV抗体阳性血清为检测阳性,24份ASFV抗体阴性血清为检测阴性,检测符合率为100％.这一研究提示制备的无标签重组K205R抗原可用于ASFV抗体检测.

为了建立猪繁殖与呼吸综合征病毒(PRRSV)接触感染模型,将易感仔猪与PRRSV人工感染仔猪在隔离器中同居饲养,每日观察记录体温变化和临床症状,定期采集血清和粪便样品,用荧光定量RT-PCR检测病毒血症和粪便排毒水平;对病死猪和试验结束迫杀猪进行剖检和肺脏组织病理学检查,并检测各器官病毒载量和组织分布.结果 显示:接触感染仔猪出现高热和PRRS症状时间分别较人工接种猪推迟2～3d;第3天起血清检测PRRSV阳性,第10天病毒血症上升至较高水平(108.6拷贝/mL);第5天起粪便检测PRRSV阳性,第7天粪便排毒量上升至较高水平(103.9拷贝/0.1g);3头接触感染仔猪分别于同居后第12天和第13天死亡,较人工接种猪推迟1～2d,病死猪的主要器官病毒载量以及剖检和肺脏显微病理变化与人工接种病死猪相似.这些结果表明,本研究建立的接触感染仔猪模型可用于PRRS疫苗免疫效果评价.

为了探明纯化标签对盘尾丝虫活化相关分泌蛋白1(activation-associated secreted protein 1,ASP1)佐剂活性的影响,将其佐剂活性区PR-1编码序列分别与类弹性蛋白多肽(elastin-like polypeptide,ELP)、ELK16自聚肽或His标签进行融合表达,用相变循环、离心洗涤和镍亲和层析进行融合蛋白纯化;将ELP与传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)VP2基因片段进行融合表达,用蛋白酶切除ELP标签后与不同标签融合PR-1免疫小鼠,两次免疫后不同时间采血分离血清,采用ELISA检测VP2特异IgG、IgG1和IgG2c滴度,用试剂盒检测免疫小鼠血清的IFN-γ、TNF-α、IL-6、IL-10浓度.结果显示,ELP-PR1、ELK-PR1、His-PR1和ELP-VP2融合蛋白在重组大肠杆菌中均获得正确表达,纯化蛋白纯度大于90％;3种标签融合PR-1均能增强免疫小鼠的抗原特异IgG、IgG1和IgG2应答,并能刺激小鼠产生IFN-γ、TNF-α、IL-6或IL-10细胞因子.在3种PR-1融合蛋白中,ELP-PR1的佐剂活性最强,ELK-PR1与不完全弗氏佐剂相当.本研究探索了不同纯化标签对PR-1免疫佐剂活性的影响,为传染性法氏囊病新型亚单位疫苗佐剂研制提供思路.

Porcine circovirus 2 (PCV2)-associated diseases affect the swine industry worldwide. Vaccination is the major tool for the disease control, but the vaccine production is hindered by lower propagation rate of PCV2 in vitro. Previous studies showed that interferons (IFNs) can increase PCV2 yield in PK-15 cells. In the present study, we constructed a Sleepy Beauty (SB) transposon vector expressing porcine IFNg gene fused with the coding sequence for immunoglobulin G Fc domain. After dilution cloning, the transposon and transposase vectors were co-transfected into PK-15 cell clones with higher permissivity to PCV2 infection. Two transgenic PK-15 cell lines, namely PK15-IFNgRan and PK15-IFNgSB which contained randomly integrated transfer vector or SB cassette without selection marker, were screened by PCR analysis. The characterization results demonstrated that the two transgenic cell lines can stably express IFNg-Fc fusion protein with potent antiviral activities. Both viral titration and quantitative PCR analyses showed that the two transgenic cell lines are highly permissive to PCV2 infection with significantly increased viral yields. These results indicate that the two transgenic PK-15 cell lines, PK15-IFNgSB in particular, can be used for PCV2 vaccine development.

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.

This study was to compare the therapeutic effect of human lysozyme( LYZ) and/or bovine interferon-γ( BoIFN-γ) expression vector( s) with antibiotics on dairy cow mastitis. BoIFN-γ c DNA was sub-cloned into the eukaryotic expression vector pcDNAK and BoIFN-γ expression in NIH 3 T3 cells was detected by immunofluorescence after transfection with the recombinant vector p CDNAK-BoIFN-γ.Through quarter base punctuation,each quarter of dairy cows with mastitis was injected two times at one-day intervals with 400μg pcDNAKLYZ,pcDNAK-BoINF-γ,pcDNAKLYZ plus pcDNAK-BoINF-γ or 100 U ampicillin plus 25μg streptomycin. For clinical and subclinical mastitis,the pcDNAKLYZ-treated group showed therapeutic efficacies of 80% and 79%,respectively. The pcDNAK-BoINF-γ-treated group showed therapeutic efficacies of 50% and 67%,respectively. The pcDNAKLYZ plus pcDNAK-BoINF-γ-treated group showed therapeutic efficacies of 100% and 84%,respectively. The antibiotics-treated group showed therapeutic efficacies of 50% and 60%,respectively,on the basis of clinical observation and/or somatic cell and total bacterial counting at day 10 after injection. These data suggested that co-injection of human LYZ and BoIFN-γ expression vectors had a higher therapeutic effect than single vector or antibiotics injection.

The clinical use of recombinant interferons (rIFNs) is limited by higher purification cost and quick clearance from circulation. Elastin-like polypeptides (ELPs) are a novel tag for recombinant protein purification and half-life extension. In this study, we evaluated the feasibility of ELP fusion for simple purification and half-life extension of recombinant porcine IFNs (rPoIFNs). After construction of five different fusion expression vectors, we optimized the conditions for soluble protein expression and purification. SDS-PAGE analysis showed that, unlike PolFN alpha-His and PolFrsl gamma-His, PoIFN alpha-ELP, ELP-PolFN alpha and PolFN alpha gamma-ELP were expressed mainly as soluble proteins at 20 degrees C. The optimal conditions for the inverse transition cycling (ITC) of three ELP fusion proteins were 2 M NaCl at 28 degrees C. After two rounds of ITC, the three ELP fusion proteins were purified to more than 90% purities, which were comparable to that of affinity-purified PoIFN alpha-His and PoIEN gamma-His. Cytopathic effect inhibition assay showed that the five rPolFNs had potent but different antiviral activities against two different viruses on two different cell types. The plasma solubility assay showed that the three ELP-fused rPoIFNs remained as soluble proteins under the physical conditions. The plasma stability of three ELP-fused rPoIFNs was significantly improved in comparison with that of PoIEN-alpha. These data suggest that ELP fusion is a feasible strategy to enhance purification and plasma stability of rPoIFNs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases affecting pig industry worldwide. Sialoadehesin (Sn) and CD163 are the two specific receptors for PRRSV infection of porcine alveolar macrophages. Our previous study showed that the soluble Sn receptor Sn4D-Fc and soluble CD163 receptor SRCR59-Fc expressed by the two recombinant adenoviral (rAd) vectors have an additive anti-PRRSV effect in vitro. In the present study, rAd-Sn4D-Fc and rAd-SRCR59-Fc were inoculated into pigs, and the efficient expression of Sn4D-Fc and SRCR59-Fc proteins was detected by ELISA. Then, PRRSV-naïve pigs were inoculated with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc before contagious infection with different PRRSV strains. Among the three rAd inoculation groups, simultaneous inoculation with the two rAd vectors provided the best protection against highly pathogenic JXA1 strain PRRSV, followed by rAd-SRCR59-Fc inoculation and rAd-Sn4D-Fc inoculation. Clinical observation and quantitative RT-PCR analyses showed that all of the double rAd-inoculated pigs (n = 9) survived from the contagious infection with highly pathogenic JXA1, JS07 or SH1705 strain PRRSV with significantly alleviated clinical scores, viremia, fecal viral emission and tissue virus loads. These data suggest that rAd-Sn4D-Fc and rAd-SRCR59-Fc can be developed further as the universal therapeutic vaccine to facilitate PRRSV eradication.

为了制备重组P128蛋白用于葡萄球菌性奶牛乳房炎治疗.将引入烟草蚀纹病毒蛋白酶(TEVp)识别位点的P128编码序列插入类弹性蛋白多肽(ELP)融合表达载体,转化大肠杆菌后用IPTG诱导融合蛋白表达,用温度敏感相变循环纯化,用TEVp活性包涵体切割融合标签,用最小抑菌浓度(MIC)和最小杀菌浓度(MBC)试验测定重组P128对奶牛乳房炎葡萄球菌抗菌活性.结果显示,融合蛋白以可溶性蛋白表达,两次相变循环纯化后的纯度达90％,产量为400 mg/mL;TEVp活性包涵体切割融合标签的效率接近100％,回收的重组P128纯度达98％,对牛源葡萄球菌的MIC和MBC分别为0.47μg/mL和1.88 μg/mL;除个别多重耐药菌株外,重组P128蛋白对多数金黄色葡萄球菌和凝固酶阴性葡萄球菌的溶(杀)作用很强.这些研究结果表明,重组P128可用于葡萄球菌性奶牛乳房炎治疗.

To establish a sandwich ELISA for quantitative detection of porcine reproductive and respiratory syndrome virus(PRRSV)soluble receptors,porcine sialoadhesin(Sn)soluble receptor Sn4D-Fc was purified from the recombinant adenovirus-transduced PK-15 cell and used as the standard antigen,mouse anti-porcine Sn serum was used as the first antibody and biotin-labeled rabbit anti-porcine Sn polyclonal antibody as the second antibody.The established sandwich ELISA was validated by detection of the soluble receptor in the serum samples of rAd-Sn4D-Fcinjected pigs.The results showed that the Sn4D-Fc standard antigen was purified to a purity of 94.3%,and was able to be recognized by anti-Sn serum.By using 5μg/mL of the first antibody and 1∶4 000 of second antibody,the detection limit of established sandwich ELISA was up to 0.4 ng/mL without cross reaction to control antigen.The Sn4D-Fc soluble receptor could be detected in the rAd-injected pigs with the highest expression level of 6.54 ng/mL and the longest duration of 15 d.These data suggested that the established sandwich ELISA was usable for quantitative detection of PRRSV soluble receptors in vitro and in vivo.