Immunization with recombinant surface antigen P50 of Babesia gibsoni expressed in insect cells induced parasite growth inhibition in dogs

This is a report of a vaccine trial directed against Babesia gibsoni infection in dogs with the use of the recombinant antigen P50. Dogs immunized with P50 showed partial protection manifested as a significantly low level of parasitemia. The results indicated that P50 is a primary vaccine candidate molecule against canine B. gibsoni infection.     

Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated B. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.     

Inhibitory effect of antiserum to surface antigen P50 of Babesia gibsoni on growth of parasites in severe combined immunodeficiency mice given canine red blood cells

The inhibitory effect of an antiserum to surface protein P50 of Babesia gibsoni on the growth of the parasite was determined with severe combined immunodeficiency mice given canine red blood cells. The antiserum to the recombinant P50 protein significantly inhibited the parasite growth, indicating that P50 might be a useful vaccine candidate.     

High-level expression and characterization of hepatitis B virus surface antigen in silkworm using a baculovirus vector.

The Bombyx mori nuclear polyhedrosis virus (BmNPV) in the silkworm was used successfully for mass production of biologically active foreign genes under the control of the polyhedrin promoter. This system was adapted for the production of large amounts of hepatitis B virus surface antigens (HBsAg). The DNA fragments coded for the middle protein, which is composed of the S protein with the pre-S2 region, were cloned, the signal protein gene of beta-IFN was added, and both were inserted into a cloning vector. After co-transfection with wild-type BmNPV, stable recombinant viruses were isolated by the limiting dilution method. Infected silkworm larvae with the recombinants expressed HBsAg at high levels (400-600 micrograms/ml). These products, consisting of two polypeptides with molecular weights of approximately 25,000 (p25) and glycosylated P25 (GP28), were purified as assembled 22-nm particles. We demonstrated that HBsAg from silkworms consists of S protein with 7 amino acids of Pre-S2.     

EV71型vP1蛋白表达、纯化及免疫原性的初步评价

目的运用Bac-to-Bac杆状病毒表达系统表达新肠道病毒EV71 vP1蛋白(EV71vP1),并对EV71 vP1蛋白的免疫原性进行初步评价。方法利用RT-PCR技术获得EV71 vP1基因,将基因序列克隆到载体pFastBac HTA,通过转座反应,与Bacmid DNA重组,重组后转染Sf9昆虫细胞。采用透射电镜观察法、SDS-PAGE、Western-blot方法对vP1蛋白进行验证,利用ELISA、微量中和抗体方法对蛋白的免疫原性进行初步评价。结果 EV71 vP1蛋白相对分子量约为33KD,表达量约10mg/L;ELISA抗体效价为1∶900;中和抗体效价1∶800。结论利用Bac-to-Bac杆状病毒表达系统成功的表达了EV71 vP1蛋白,并对其免疫原性做了初步评价,为今后EV71 vP1亚单位疫苗的研究提供参考。     

Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector

Simian virus 40 (SV40) large T and small t antigens were synthesized in insect cells using the baculovirus Autographa californica as an expression vector. A recombinant virus containing a genomic copy of the SV40 early region expressed high levels of small t antigen but only low levels of large T antigen. However, very high levels of T antigen synthesis were observed when viruses were constructed with a cDNA copy of the large T antigen mRNA. Insect cells were capable of modifying T antigen by phosphorylation, palmitylation, glycosylation, and oligomerization. Functional assays demonstrated that the origin-specific DNA binding, ATPase, and helicase activities of insect cell-derived T antigen were comparable to T antigen synthesized in mammalian cells. Use of the baculovirus vector system to produce T antigen should facilitate future investigations requiring large quantities of T antigen.     

High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis

The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies in B. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence of B. equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.     

Characterization of pseudorabies virus neutralization antigen glycoprotein gIII produced in insect cells by a baculovirus expression vector.

The gene encoding the complete glycoprotein of pseudorabies virus (PRV, Yamagata-S81 strain glycoprotein gIII) has been inserted into the baculovirus transfer vector pAcYM1S derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). A Spodoptera frugiperda cell line, SF21AE, was efficiently co-transfected with the transfer vector containing the gIII gene and AcNPV DNA by cationic liposomes (Lipofectin). The gene was placed under the control of the AcNPV polyhedrin promoter and expressed to high levels by the derived recombinant virus using SF21AE. Three polypeptides of different molecular weight were expressed. The principal products were glycosylated and transported to the cell surface. The smallest product was not glycosylated. Despite their lower molecular weight, it has been established that the antigenic properties of the peptides were conserved by comparison with those of the authentic glycoprotein gIII of PRV. Immunogenicity of the expressed products was also demonstrated. Intraperitoneal injection of expressed gIII induced neutralizing antibodies in mice. The results have raised the possibility that the protein expressed by baculovirus recombinant may be used to analyze biologically functional sites, develop a subunit vaccine and diagnostic antigens.     

Purity, antigenicity and immunogenicity of the hepatitis B surface antigen purified by five different methods

Five published methods for the purification of HBsAg from plasma were compared for specific activity (SA), degree of purification, and yield. The SA value was determined by dividing the reciprocal of the end point dilution per milliliter as determined using a commercial radioimmunoassay (AUSRIA II; Abbott Laboratories, North Chicago, IL) by the protein concentration quantitated by the Lowry method. HBsAg purified by two consecutive isopycnic ultracentrifugation separations in KBr and one rate-zonal separation in sucrose using a zonal rotor (Ti-14, Beckman, Palo Alto, CA) yielded a preparation which gave the highest SA value, degree of purification and yield as compared to four other methods. Each purified preparation was adsorbed to alum adjuvant and injected into mice to determine the immunogenic dose at which 50% of the animals elicited an anti-HBs response (ID50). The zonal rotor method resulted in the lowest ID50 value (365 ng/ml) supporting the highest SA value. Furthermore, SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed that this preparation had the greatest number of HBsAg-specific polypeptides (N = 7) and the fewest contaminating polypeptides (N = 5). The contaminating proteins were identified as alpha-2-macroglobulin, heavy chains of IgG and IgM, immunoglobulin kappa chain, and albumin.     

High-Level Expression of Truncated Surface Antigen P50 of Babesia gibsoni in Insect Cells by Baculovirus and Evaluation of Its Immunogenicity and Antigenicity

Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10⁸ cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.     

Epstein-Barr病毒膜抗原在昆虫细胞中的表达

目的　探索影响EpsteinBarr病毒膜抗原(EBVMA)在昆虫细胞中表达的因素。方法　携有截去穿膜区序列的EBVMA基因的重组昆虫杆状病毒感染不同生长阶段及生长条件的昆虫细胞,应用免疫打点法及MasterImagineVDS影像摄录分析系统测定目的蛋白的表达量。结果　重组病毒感染不同生长阶段的细胞,MA表达量不同。处于对数生长早期的细胞表达量明显高于处于对数生长晚期及静止期的细胞。将不同生长阶段的细胞补充新鲜培养液,MA的表达量在原基础上有所提高。进一步研究表明,在感染重组病毒之前,更换新鲜培养液,同时在感染过程中补充葡萄糖,L谷氨酰胺等营养物质,会大大提高MA表达量。结论　细胞培养液中营养物质的消耗和代谢产物的蓄积是影响EBVMA在昆虫细胞中表达的主要因素。     

谷氨酸脱羧酶65重组杆状病毒表达载体的构建及其在昆虫细胞中的表达

目的构建谷氨酸脱羧酶GAD65基因的杆状病毒重组供体质粒,包装重组GAD65的杆状病毒,感染昆虫细胞进行表达。方法先将已克隆的GAD65 cDNA与线性化的pFASTBACHTb进行连接,使pFASTBACHTb-GAD65与DH10BAC感受菌进行转座重组,利用抗性及蓝白斑筛选重组Bacmid/GAD65克隆,提取Bacmid/GAD65转染昆虫细胞Sf 9包装杆状病毒,利用病毒感染Sf9细胞进行蛋白表达,通过免疫荧光、SDS PAGE和Western blot检测表达情况。结果获得了重组GAD65的杆状病毒,细胞能表达出与GAD65单抗结合的蛋白,相对分子质量为65kDa左右,细胞可直接分泌GAD65蛋白至上清。结论GAD65能在昆虫细胞中获得良好表达为研究GAD65的作用及免疫治疗奠定基础,。     

Immunization with Recombinant Surface Antigen P50 of Babesia gibsoni Expressed in Insect Cells Induced Parasite Growth Inhibition in Dogs

This is a report of a vaccine trial directed against Babesia gibsoni infection in dogs with the use of the recombinant antigen P50. Dogs immunized with P50 showed partial protection manifested as a significantly low level of parasitemia. The results indicated that P50 is a primary vaccine candidate molecule against canine B. gibsoni infection.     

Ⅱ型单纯疱疹病毒糖蛋白D在杆状病毒表达系统中的表达、纯化及免疫效果评价

目的:利用Bac-to-Bac杆状病毒表达系统对Ⅱ型单纯疱疹病毒(HSV-2)糖蛋白D(gD2)进行表达,纯化后评价其免疫效果,以探索HSV-2重组亚单位疫苗的研制。方法:提取HSV-2DNA作为模板,用聚合酶链式反应(PCR)扩增gD2基因,克隆至pFastBac HTA载体中,利用载体中的His基因标记gD2,通过Bac-to-Bac杆状病毒表达系统表达His-gD2融合蛋白,表达产物进行SDS-PAGE和Western-blot检验,用镍柱进行纯化,免疫小鼠评价其免疫原性。结果:HSV-2gD2在昆虫细胞Sf9中得到特异性表达,纯化后的重组蛋白诱导小鼠体内产生高水平的IgG抗体及中和抗体。结论:gD2基因在Bac-to-Bac杆状病毒表达系统中获得表达,表达产物表现出良好的免疫原性及中和活性,为发展HSV-2亚单位疫苗奠定了基础。     

转染双启动子杆状病毒表达载体在昆虫细胞共表 …

目的 通过体外表达ｒｈＩＬ－１２,以供研究其在体内外的生物学效应。方法 外周血单个核细胞（ＰＢＭＣ）、粘时细胞和人口腔表皮样癌细胞ＫＢ分别经ＳＡＣ（含Ａ蛋白的金黄色葡萄球菌ＣｏｗａｎⅠ菌株）、ＩＦＮ－γ＋ＬＰＳ和ＰＤＢｕ（１２、１３－二丁酸佛波酯）刺激,以ＧＩＴ（异硫氰酸胍）一步法提取细胞总ＲＮＡ,经ＲＴ－ＰＣＲ技术获得ＩＬ－１２ Ｐ４０和Ｐ３５ ｃＤＮＡ,将两条基因分别插入双启动子杆状病毒转染载     

Expression of Epstein-Barr virus nuclear antigen 2 in insect cells from a baculovirus vector.

The open reading frame encoding the Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been expressed in a recombinant baculovirus vector. The resulting product migrates with the same apparent molecular weight as EBNA-2 from latently infected or converted B cell lines. Rabbit antisera derived from the innoculation of this material immunoprecipitated EBNA-2 from cell extracts of EBV-containing cells. The high level of protein expression obtained in insect cells stands in sharp contrast to that seen in a number of mammalian cell lines using a variety of promoters including the endogenous EBNA-2 promoter, the Moloney MuLV LTR, the murine immunoglobulin heavy chain promoter, the human cytomegalovirus immediate early promoter, and the adenovirus major late promoter.     

Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system.

The baculovirus expression vector p36C was used to express in cells of the insect Spodoptera frugiperda fragment C of tetanus toxin under the control of the strong polyhedrin promoter. Fragment C was expressed intracellularly at a high level and was soluble, allowing it to be purified by affinity chromatography with monoclonal antibody TT08. Purified fragment C from baculovirus was used to immunize mice and was shown to successfully prevent the symptoms of tetanus following a toxin challenge. The ganglioside-binding properties of baculovirus-derived fragment C were compared with those of intact tetanus toxin and native fragment C and were found to be dissimilar.     

Characterization of Toxoplasma gondii SAG2 expressed in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats.