麻疹病毒血凝素和融合蛋白基因的克隆与表达及其重组蛋白的免疫学评价

采用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）的方法从麻疹病毒Ｅｄｍｏｎｓｔｏｎ株基因组中扩增出血凝素Ｈ基因和融合蛋白Ｆ基因，并利用转移质粒将这两个基因分别重组到杆状病毒多角体蛋白启动子（ＰＨ）控制之下，获得重组病毒ｖＢＭＶＨ和ｖＢＭＶＦ。重组杆状病毒感染Ｓｆ９昆虫细胞，表达的重组蛋白分别具有血凝、血溶活性。红细胞吸附抑制试验、免疫印迹、酶联免疫试验结果显示重组蛋白在生物学、生化学特性与天然蛋白类似，并能被天然麻疹免疫血清（人、鼠、兔）所识别。动物实验表明，重组蛋白具有诱生中和抗体的能力。重组血凝素免疫血清还具有血凝抑制抗体活性。这些结果说明杆状病毒－昆虫细胞体系表达的麻疹病毒血凝素和融合蛋白具有较好的生物学活性及免疫原性和抗原性。     

Expression and cellular distribution of baculovirus-expressed bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) sequences

Summary.  Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus) – insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences. Received March 2, 1998 Aaccepted June 16, 1998     

Expression of the fusion glycoprotein of human parainfluenza type 3 virus in insect cells by a recombinant baculovirus and analysis of its immunogenic property

The fusion (F) glycoprotein of human parainfluenza type 3 (PI3) virus was produced in insect cells using a baculovirus expression vector (pAcYM1). The recombinant glycoprotein was identified by its reactivity with specific monoclonal and polyclonal antibodies and showed an apparent molecular mass of 70 kDa. Although the fusion protein was found on the infected cell surface, it did not appear to be proteolytically cleaved to F1 and F2 subunits. Immunization of hamsters with the recombinant protein elicited antibody which neutralized infectivity and blocked fusion of virus-infected cells. The protective response to challenge infection of immunized hamsters was similar to that observed with affinity purified F from PI3 virus (Ray et al., J. Virol. 62, 783-787, 1988).     

狂犬病病毒糖蛋白和核蛋白基因的克隆及其重组蛋白在昆虫细胞中的表达

目的从国内狂犬病疫苗ａＧ株中克隆狂犬病病毒糖蛋白（ＧＰ）基因和核蛋白（ＮＰ）基因，应用杆状病毒－昆虫细胞表达系统使其在昆虫细胞中表达。方法从狂犬病病毒感染细胞上清液中提取病毒ＲＮＡ，应用ＲＴ－ＰＣＲ方法扩增ＧＰ基因和ＮＰ基因。扩增的基因与转移质粒连接并转化大肠杆菌，得到重组转移质粒。将其与野生杆状病毒（ＡｃＭＮＰＶ）线性ＤＮＡ共转染Ｓｆ９昆虫细胞，通过有限稀释法筛选含有ＧＰ基因和ＮＰ基因重组病毒。初步检测了重组蛋白的抗原性。结果用ＲＴ－ＰＣＲ方法扩增得到ＧＰ基因和ＮＰ基因，通过重组转移后重组病毒感染的细胞经免疫印染实验表明可分别表达ＧＰ和ＮＰ重组蛋白。重组蛋白的分子量分别为５８×１０３和５３×１０３。用重组病毒感染的细胞免疫小鼠后可诱导动物产生特异性抗体。结论可以应用杆状病毒－昆虫细胞表达系统表达狂犬病病毒ＧＰ和ＮＰ重组蛋白，为开发基因工程化狂犬病疫苗提供有意义的资料     

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D

Summary The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirusAutographa california nuclear polyhedrosis virus (AcNPV). After co-transfection ofSpodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.     

抗甲肝病毒人源基因工程全抗体分子在杆状病毒中的表达

目的 探讨人源抗甲型肝炎病毒全抗体分子在杆状病毒中的表达。方法 将获得的人源抗甲肝病毒中和性抗体Fab段基因克隆入含信号肽及Fc的杆状病毒表达载体中并在杆状病毒细胞中表达。结果 获得了中和性人源抗甲肝病毒全抗体分子的表达产物并进行了纯化，轻重链表达产物位置大小正确，HAFc16抗体能与具有中和活性的鼠抗甲肝病毒单克隆抗体产生竞争抑制反应，并能在体外中和甲肝病毒，另一株HAFc78抗体同样具有体外中和甲肝病毒的活性，但系抗不同位点的抗体。结论 获得的人源抗甲肝病毒全抗体分子表达产物具有很好的体外中和甲肝病毒的活性，且为抗不同位点的抗体，为这些抗体的进一步开发及应用打下了基础，为防止甲型肝炎暴发流行提供应急措施。     

Hepatitis C virus glycoprotein E2 binding to CD81: the role of E1E2 cleavage and protein glycosylation in bioactivity

The hepatitis C virus glycoproteins E1 and 2 have been expressed using recombinant baculoviruses following fusion to the carrier protein glutathione S-transferase (GST). Proteins were expressed singly and as an E1E2 polyprotein with and without an N-terminal affinity tag. Expression of the E1E2 polyprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutathione affinity chromatography. Baculovirus expressed E2 and a purified GST-E1E2 protein bound to the second extracellular loop of CD81 (EC2), a reported ligand for the molecule, but not to a truncated derivative of CD81 consisting of only the central domain of the loop. Purified GST-E2, however, failed to bind to CD81 suggesting a requirement for a free E2 amino terminus for biological activity. The binding to CD81 by baculovirus expressed E2 protein was comparable to that observed for E2 derived from mammalian cells when detected by a monoclonal antibody sensitive to protein conformation. Furthermore, E2 protein expressed in insect cells in the presence of N-butyldeoxynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with E1 and bound to CD81-EC2 similarly to untreated protein. Together these data suggest that although hyperglucosylation of E2 does not have a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required.     

Nipah virus glycoprotein: production in baculovirus and application in diagnosis

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was cloned into the pFastBac HT vector, and the fusion protein to His-tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negatives samples. The data show that the recombinant G protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-based NiV ELISA compared to an ELISA using whole virus antigen is the use of a single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly.     

EBV-LMP2蛋白的表达和初步纯化

目的 应用杆状病毒表达载体表达并纯化EB病毒LMP2蛋白.方法 利用杆状病毒Bac-to-Bae杆状病毒表达系统,将EBV-LMP2基因插入到质粒pFastBacTMHT B中,获得携带EBV-LMP2基因的重组杆状病毒Bac-LMP2.重组病毒感染Sf-9细胞,表达N端携带6个组氨酸(6 X His)的LMP2融合蛋白His-LMP2.经镍离子亲和层析纯化,获得纯化蛋白.结果 SDS-PAGE及Western-Blot检测表达的蛋白相对分子质量大小与预计结果一致.HPLC分析纯化后蛋白的纯度可达86%.结论 利用杆状病毒表达系统表达EB病毒LMP2基因并经过初步纯化后,可以获得较好纯度的LMP2蛋白.     

EBV-LMP2蛋白的表达和初步纯化

目的 应用杆状病毒表达载体表达并纯化EB病毒LMP2蛋白.方法 利用杆状病毒Bac-to-Bae杆状病毒表达系统,将EBV-LMP2基因插入到质粒pFastBacTMHT B中,获得携带EBV-LMP2基因的重组杆状病毒Bac-LMP2.重组病毒感染Sf-9细胞,表达N端携带6个组氨酸(6 X His)的LMP2融合蛋白His-LMP2.经镍离子亲和层析纯化,获得纯化蛋白.结果 SDS-PAGE及Western-Blot检测表达的蛋白相对分子质量大小与预计结果一致.HPLC分析纯化后蛋白的纯度可达86%.结论 利用杆状病毒表达系统表达EB病毒LMP2基因并经过初步纯化后,可以获得较好纯度的LMP2蛋白.     

EBV-LMP2蛋白的表达和初步纯化

目的 应用杆状病毒表达载体表达并纯化EB病毒LMP2蛋白.方法 利用杆状病毒Bac-to-Bae杆状病毒表达系统,将EBV-LMP2基因插入到质粒pFastBacTMHT B中,获得携带EBV-LMP2基因的重组杆状病毒Bac-LMP2.重组病毒感染Sf-9细胞,表达N端携带6个组氨酸（6 X His）的LMP2融合蛋白His-LMP2.经镍离子亲和层析纯化,获得纯化蛋白.结果 SDS-PAGE及Western-Blot检测表达的蛋白相对分子质量大小与预计结果一致.HPLC分析纯化后蛋白的纯度可达86%.结论 利用杆状病毒表达系统表达EB病毒LMP2基因并经过初步纯化后,可以获得较好纯度的LMP2蛋白.     

马传染性贫血病毒囊膜蛋白GP90的表达及其免疫效果研究

目的：探索马传染性贫血病毒（EIAV）野毒株（强毒，LN）和疫苗毒株（弱毒，DLV）的囊膜蛋白GP90作为鉴别诊断试剂的效果及基因工程疫苗的可能性。方法：将中国EIAV疫苗株（DLV）及其亲本毒株辽毒株（LN）接种于驴外周血白细胞培养物，提取前病毒DNA，并以其为模板，经聚合酶链法（PCR）扩增出LN和DLV的GP90片段，在Bac-to-Bac杆状病毒表达系统表达。表达的GP90蛋白经金属离子亲和层析（IMAC）纯化后免疫小鼠，ELISA法检测抗EIAV抗体，中和试验检测中和抗体活性。同时对DLV和LN GP90的核苷酸与氨基酸进行比较。结果：DLV和LN GP90核苷酸序列的同源性为95．3％，氨基酸序列的同源性为87.7％。未加佐剂组抗体滴度为800，佐剂组抗体滴度达1600;不加佐剂组的中和抗体滴度在40－80之间，加佐剂组中和抗体滴度在80－160之间。结论：成功构建了分别表达EIAV野毒株LN和疫苗毒株DLV囊膜蛋白GP90的重组杆状病毒，大量表达的蛋白纯化后纯度较好，该纯化产物在小鼠体内可激发良好的体液免疫应答。     

Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins

Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.     

Cell-based analysis of Chikungunya virus membrane fusion using baculovirus-expression vectors

Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion.     

HIV 外膜糖蛋白 gp120 和 gp41 在昆虫细胞中表达及其免疫学特性的评价

将编码完整ｇｐ１２０和完整ｇｐ４１的基因分别克隆到杆状病毒转移质粒中。使用重组转移质粒与野生杆状病毒（ＡｃＮＰＶ）ＤＮＡ共转染Ｓｆ９昆虫细胞，经挑选获得分别带有编码ｇｐ１２０和ｇｐ４１基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中分别表达了ＨＩＶ外膜糖蛋白ｇｐ１２０和ｇｐ４１。其重组蛋白的分子量分别为１２０×１０３和４１×１０３。此重组糖蛋白在免疫荧光、免疫印染和酶联免疫实验中都能被ＨＩＶ阳性血清所识别。动物免疫实验表明此重组糖蛋白能诱导很强的特异性抗体产生。     

单纯疱疹病毒1型糖蛋白D主要抗原表位区的表达、纯化及鉴定

 目的 原核表达单纯疱疹病毒1型(HSV-1) 糖蛋白D(gD)主要抗原表位区,纯化融合表达蛋白并对其进行鉴定。方法 用Lasergene7.0中Protean软件对HSV-1 gD氨基酸序列进行抗原表位预测和分析,筛选出gD主要抗原表位区(gD MED),人工合成该区域cDNA序列,构建原核表达载体pET-GST-gD MED;将重组质粒转化大肠杆菌(E.coli)BL21(DE3) pLysS,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,通过GST?Bind 纯化试剂盒对gD MED融合蛋白进行纯化;Western blot对gD MED融合蛋白的免疫活性进行分析。结果 HSV-1 gD在266~394区域富含抗原表位,人工合成了该区域的cDNA序列,并成功构建了原核表达载体pET-GST-gD MED;gD MED融合蛋白主要以可溶形式表达,分子质量约为45 ku,纯度能达95%;Western blot结果显示,gD MED融合蛋白能与感染HSV-1患者的血清发生特异结合。结论 成功表达和纯化了具有良好抗原性的HSV-1 gD MED融合蛋白,为HSV免疫诊断试剂盒和基因工程疫苗的研制奠定了基础。     

Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies

The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.     

Expression of the X protein of hepatitis B virus in insect cells using recombinant baculoviruses

The baculovirus system was used to express the X protein of human hepatitis B virus (HBV). The X open reading frames (X ORFs) from cloned viral DNA of the HBV subtypes ayw and adr were introduced into the genome ofAutographa californica nuclear polyhedrosis virus (AcNPV). The HBV-DNA of subtype adr derived from a hepatocellular carcinoma contains an X ORF and a 5 extended preX/X ORF, which were both used to construct X recombinant baculoviruses. Infection of Sf9 insect cells with these recombinant viruses yielded large amounts of the respective X proteins. They were identified by a set of mouse monoclonal antibodies directed against different epitopes of the ayw X protein using immunoblotting techniques. A subpopulation of the X protein expressed is modified, thus raising the molecular weight from the expected size of 17 kD to 21 kD. Indirect immunofluorescence and immunoelectron microscopy was performed to characterize the subcellular distribution of the X protein expressed in Sf9 cells. Data are presented that it accumulates as large globular structures within the cytoplasm and the nucleus of the infected cells.     

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.