Properties of recombinant hepatitis B vaccine

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

Recombinant retroviral DNA yielding high expression of hepatitis B surface antigen

A genomic fragment of hepatitis B virus encoding the surface antigen (HBsAg) was inserted into the proviral genome of Moloney mouse sarcoma virus (MSV), obtained from the mouse cell line G8 -124. The recombinant DNA was introduced into NIH 3T3 mouse fibroblasts. Cells, morphologically transformed by the oncogene of MSV (v-mosM) were selected, established as cell lines and tested for expression of HBsAg. An expression level of up to 4.5 micrograms/10(7) cells/day was detected.     

Intradermal vaccination of adults with three low doses (2 micrograms) of recombinant hepatitis B vaccine. II. Persistence of immunity and induction of immunologic memory

Of the 110 dentists who had presented seroconversion 50 days after the intradermal application of three 2 micrograms doses of the Belgian recombinant vaccine against hepatitis B (HB), administered eight years before at an interval of one month between the 1st and 2nd doses and of five months between the 2nd and 3rd doses, 51 were included for the assessment of the persistence of immunity. None of the dentists had hepatitis or had received HB vaccine during this period. All subjects were submitted to serological tests for the detection of the following markers of hepatitis B virus (HBV) infection: HBsAg, anti-HBc, HBeAg, anti-HBe, and anti-HBs, with no HBsAg, anti-HBc, HBeAg or anti-HBe being detected. A microparticle enzyme immunoassay (MEIA) revealed the presence of anti-HBs at protective titers (> or = 10 mIU/ml) in 42 dentists (82.4%), with the anti-HBs titer being higher than 100 mIU/ml in 36 of them (70.6%) (good responders), between 10 and 100 mIU/ml in 6 (11.8%) (poor responders), and lower than 10 mIU/ml in 9 (17.6%) (non-responders). According to clinical data and serological tests, none of the dentists had presented disease or latent HBV infection during the eight years following the first vaccination. A 2 micrograms booster dose was administered intradermally to eight dentists with anti-HBs titers lower than 10 mIU/ml (non-responders) and to six dentists with titers ranging from 10 to 100 mIU/ml (poor responders); the determination of anti-HBs one month later demonstrated the occurrence of seroconversion in the eight non-responders and an increase in anti-HBs titer in the six poor responders. In summary, the present results demonstrated the prolonged persistence of protection against HBV infection and the development of immunologic memory provided by vaccination against HB--with intradermal application of three 2 micrograms doses of the Belgian recombinant vaccine at 0, 1, and 6 months--carried out eight years before in 51 dentists.     

Improving the recovery of clarification process of recombinant hepatitis B surface antigen in large-scale by optimizing adsorption-desorption parameters on Aerosil-380

Abstract

In the downstream process of recombinant hepatitis B surface antigen (rHBsAg), nano-colloidal silica adsorbent (Aerosil-380) is one of the possible methods to separate the antigen from other main impurities partially. The current study aimed to maximize the adsorptive capacity of Aerosil-380 as well as rHBsAg recovery for large-scale production of recombinant hepatitis B vaccine. The experimental design methodology was used to optimize the eight critical parameters influencing the efficiency, rHBsAg recovery, of the adsorption-desorption process in the lab-scale. These examined parameters were the adsorption–desorption temperature, pH, contact time, agitation speed, antigen concentration, and desorption buffer. Under optimal condition, the maximum adsorption capacity of Aerosil-380 was equal to 3333?μg.g^?1 (rHBsAg/adsorbent), and we could recover about 95% of rHBsAg with purity of 54% (rHBsAg/total protein) in the lab scale. Using the optimum parameters for rHBsAg clarification process in large-scale by Aerosil-380, we recovered about 78% of rHBsAg with 43% purity. Based on the obtained experimental data, Langmuir adsorption isotherm and pseudo-first-order kinetic model provide the best correlations of experimental data for the adsorbent. Findings of this study significantly increase the recovery of clarification process of rHBsAg in large-scale compared to previous reports.     

Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation.

The need for an accurate, fast and reliable analysis of carbohydrate test is crucial for numerous biological processes. In that sense, anthrone-sulfuric acid assay is one of the most efficient quantification techniques successfully applied to carbohydrate determination. In this paper, a sensitive and accurate anthrone-sulfuric acid microplate assay was developed and validated for the quantitative estimation of yeast carbohydrates in the production of hepatitis B virus surface antigen, and the main component of the recombinant vaccine HEBERBIOVAC HB. A response surface methodology was applied to design and optimize the assay in order to maximize the differences on the expected effect and to minimize the number of experiments. The proposed method was linear over the concentration range from 10 to 120 microg/mL for glucose, with values for the coefficient of determination >0.99. Intra- and inter-assay variation coefficient ranged between 0.45-4.79% and 2.48-8.94%, respectively. The Student t-test used in the interference study, revealed good parallelism among curves (T(obs)< or =T(0.05)), which indicates the lack of interference in the working range. Yields obtained in accuracy test for two concentration levels varied between 90 and 105%, confirming the assay's reliability. In conclusion, the validated method, which has successfully been used for the process control monitoring of several samples generated from the production of hepatitis B vaccine, allows the quality and purity of the final product.     

特异性表达siRNA的新型乙肝核酸药物的研究

旨在研究靶向于HBsAg的肝脏特异性siRNA,实现核酸药物高靶向性、高特异性基因治疗乙肝。通过重组PCR的方法构建靶向于HBsAg的肝脏特异性表达的siRNA的核酸药物。将目标载体转染HepG2 2.2.15细胞,用ELISA的方法测定转染细胞培养液内HBsAg的含量。PCR鉴定和测序确定重组PAAV-MCS载体PApoE1-ApoE2-hAAT-siHBsAg-U1 3’Box构建成功。重组质粒对HepG2 2.2.15细胞分泌的HBsAg所产生的抑制作用自转染后5 d达到高峰,抑制率达到(30±0.28)%(P<0.01),siRNA特异性表达单元能显著降低HepG2 2.2.15细胞HBV DNA的拷贝数。成功构建的肝脏特异性、siRNA高表达的核酸药物可以有效地抑制HBV基因的表达和复制,为乙肝的进一步治疗研究做了关键性工作。     

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells

Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.     

Results of the examination of patients with viral hepatitis for HBsAg in Kashka-Darya Province, Uzbek SSR

The practical use of the reverse passive hemagglutination test for the examination of viral hepatitis patients revealed the presence of HBsAg, on the average, in 28.4 +/- 0.95% of cases. This antigen occurred most frequently among the patients aged up to 1 year (42.8 +/- 4.18%), as well as in the second quarter of the year (33.3 +/- 2.3%). The proportion of patients with hepatitis B in the total number of patients with viral hepatitis was 20%.     

Controlled fed-batch fermentation of recombinant Saccharomyces cerevisiae to produce hepatitis B surface antigen

We have performed controlled fed-batch fermentation experiments to compare the production level of hepatitis B surface antigen (HBsAg) by recombinant yeast Saccharomyces cerevisiae strains (YNN27/pYBH-1, YNN27/ p2mu-S11, YNN27/pDCB-S2) containing plasmid vector with alcohol dehydrogenase (ADH1), acid phosphatase (PHO5), and glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, respectively. Yeast cell concentrations of 15-35 g dry cell weight/L were obtained. By limiting phosphorous concentration, HBsAg expression level for the YNN27/p2mu-S11 strain with inducible PHO5 promoter reached 0.2-0.3 mg/L. By controlling nutrient addition rate and dissolved oxygen concentration, HBsAg concentrations of 3-10 mg/L were achieved in 60-70 h fermentation using the YNN27/pDCB-S2 strain with the constitutive GPD promoter.     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid‐envelope virus‐like particles

This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid‐envelope virus‐like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving ～70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of ～80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions (～400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low‐shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two‐fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

重庆地区乙肝血清特殊模式患者HBV基因分型及临床自然病程分析

目的了解重庆地区乙肝病毒（HBV）血清学标志物为特殊模式的HBV感染患者病毒基因型的分布情况,分析其临床特征及自然病程。方法从1000例HBV感染者中检测到48例乙肝病毒血清学标志物为特殊模式的患者（HBsAg与抗一HBs同时阳性,HBeAg与抗一HBe同时阳性）。采用巢式聚合酶链式反应（nPCR）对特殊模式患者的HBV进行基因分型,同时对两组特殊模式患者的临床资料和HBV感染的自然史进行分析。结果48例乙肝病毒血清学标志物为特殊模式的HBV感染者中,36例患者HBsAg与抗-HBs同时阳性,12例患者HBeAg与抗-HBe同时阳性。HBeAg＋／抗-HBe＋患者组的年龄较HBsAg＋／抗-HBs＋患者组的小（P〈0．05）。HBsAg＋／抗-HBs＋患者中,3例（8．3％）为B2亚型,12例（33．3％）为c2亚型,21例（58．4％）未分型;HBeAg＋／抗-HBe＋患者中,8例（66．7％）为B2亚型,1例（8．3％）为c2亚型,3例（25．0％）未分型,两组在HBV基因型的分布上差异具有统计学意义（Y2=17．44,P〈0．05）。在HBsAg＋／抗-HBs＋患者中,2例（4．2％）处于免疫清除期,14例（29．2％）处于低复制期,7例（14．6％）处于再活动期。HBeAg＋／抗-HBe＋患者中,5例（10．4％）处于免疫清除期。两组在HBV感染的自然病程中的分布差异具有统计学意义（X2=18．26,P〈0．05）。结论重庆地区乙肝病毒血清学标志物为特殊模式的慢性HBV感染者中,HBeAg与抗-HBe同时阳性的HBV感染者中B2亚型为优势基因型;HBsAg与抗-HBs同时阳性的HBV感染者中,HBV基因型以C2亚型为主。     

Evidence for hepatitis C viral infection in patients with primary hepatocellular carcinoma.

In testing for antibodies to the hepatitis C virus (anti-HCV) in 112 patients with primary hepatocellular carcinoma, 10 of 33 white patients (30%) and 15 of 79 Asian patients (19%) had a positive response to the antibody. The antibody profile to individual hepatitis C viral antigens and the presence of circulating hepatitis C viral RNA were determined in the 25 patients. The anti-HCV antibodies most frequently detected were toward the antigens from the core (C22) and NS3 regions. Serum hepatitis C viral RNA was present in 17 of the 25 patients (68%), and these patients tended to have serum levels of alanine and aspartate aminotransferases higher than those patients without viremia (136 +/- 22 U per liter versus 64 +/- 11 U per liter and 161 +/- 26 U per liter versus 79 +/- 14 U per liter, respectively, both P < .05). Of the 15 Asian patients with hepatocellular carcinoma and anti-HCV, 4 (27%) had coexisting hepatitis B surface antigen (HBsAg) and 13 (87%) had antibodies to either hepatitis B core or surface antigen. Of the 10 white patients with anti-HCV, however, only 1 (10%) had hepatitis B virus antibodies (P < .01). Among 4 Asian patients with coexisting anti-HCV and HBsAg, 1 was found to have serum hepatitis B viral DNA and the other 3 had hepatitis C viral RNA. A history of blood transfusion was obtained from 12 of the 25 patients with anti-HCV (48%); 20 (80%) had coexisting cirrhosis. Our findings support the hypothesis that hepatitis C virus is an important etiologic agent in the development of primary hepatocellular carcinoma in both white and Asian patients in the United States.     

Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.     

新生儿国产重组(酵母)乙型肝炎疫苗免疫效果考核

为了考核新生儿接种国产重组(酵母)乙型肝炎(乙肝)疫苗后的免疫效果,并与血源乙肝疫苗效果比较。对1997年出生并接种重组(酵母)乙肝疫苗的新生儿隔年随访一次,采血检测乙肝病毒表面抗原(HBsAg),乙肝病毒表面抗体(抗-HBs)和乙肝病毒核心抗体(抗-HBc),1998年以后对乙肝免疫人群开展急性乙肝发病监测。显示五年期间3次随访检测HBsAg阳性率平均为1.5%,较免前本底的HBsAg阳性率呈较大幅度下降,疫苗保护率为83%(95%可信区间为76.97%~89.02%),无论母亲HBsAg阳性或阴性,使用不同乙肝疫苗的儿童HBsAg阳性率没有统计学差异。接受重组(酵母)乙肝疫苗免疫的对象中,无一例急性乙肝病例报告。重组(酵母)乙肝疫苗有较好的近期保护效果和免疫原性,与以前使用血源乙肝疫苗效果相当。     

Effect of hematocrit reduction on hormonal and metabolic responses to exercise

We wished to determine the effect of a 25% hematocrit reduction on glucoregulatory hormone release and glucose fluxes during exercise. In five anemic dogs, plasma glucose fell by 21 mg/dl and in five controls by 7 mg/dl by the end of the 90-min exercise period. After 50 min of exercise, hepatic glucose production (Ra) and glucose metabolic clearance rate (MCR) began to rise disproportionately in anemics compared with controls. By the end of exercise, the increase in Ra was almost threefold higher (delta 15.1 +/- 3.4 vs. delta 5.2 +/- 1.3 mg X kg-1 X min-1) and MCR nearly fourfold (delta 24.6 +/- 8.8 vs. delta 6.5 +/- 1.3 ml X kg-1 X min-1). Exercise with anemia, in relation to controls resulted in elevated levels of glucagon [immunoreactive glucagon (IRG) delta 1,283 +/- 507 vs delta 514 +/- 99 pg/ml], norepinephrine (delta 1,592 +/- 280 vs. delta 590 +/- 155 pg/ml), epinephrine (delta 2,293 +/- 994 vs. delta 385 +/- 186 pg/ml), cortisol (delta 6.7 +/- 2.2 vs. delta 2.1 +/- 1.0 micrograms/dl) and lactate (delta 12.1 +/- 2.2 vs. delta 4.2 +/- 1.8 mg/dl) after 90 min. Immunoreactive insulin and free fatty acids were similar in both groups. In conclusion, exercise with a 25% hematocrit reduction results in 1) elevated lactate, norepinephrine, epinephrine, cortisol, and IRG levels, 2) an increased Ra which is likely related to the increased counterregulatory response, and 3) we speculate that a near fourfold increase in MCR is related to metabolic changes due to hypoxia in working muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen

An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP–HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3.     

Tandem mass spectrometric determination of 11-dehydrothromboxane B2, an index metabolite of thromboxane B2 in plasma and urine

11-Dehydrothromboxane B2 is one of the major enzymatic metabolites of thromboxane B2 (TXB2), a biologically inactive product of thromboxane A2. The short half-life of thromboxane A2 and ex vivo production of thromboxane B2 by platelet activation make these prostanoid metabolites inappropriate as indices of systemic thromboxane biosynthesis, whereas 11-dehydro-TXB2 has been shown to reflect the release of thromboxane A2 in the human blood circulation. Analysis of 11-dehydro-TXB2 in plasma and urine was performed by gas chromatography-mass spectrometry-mass spectrometry using the chemically synthesized tetradeuterated compound as an internal standard. The high selectivity of triple-stage quadrupole mass spectrometry (tandem mass spectrometry) considerably facilitates sample purification as compared to single quadrupole mass spectrometric determination. Plasma concentrations in five healthy male volunteers were in the range 0.8-2.5 pg/ml. Urinary excretion of 11-dehydro-TXB2 was higher than that of 2,3-dinor-TXB2: 1.2 +/- 0.36 micrograms/24 h vs 0.53 +/- 0.33 micrograms/24 h (n = 5). Thus 11-dehydro-TXB2 appears at present to be the best index metabolite of systemic TXA2 activity in plasma as well as in urine.     

Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification

Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 μg/ml in the haemolymph. The scFv-gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. Judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials.