Expression of equine interleukin-18 by baculovirus expression system and its biologic activity

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.     

虎源流感病毒HA 基因的系统发生分析及其在杆状病毒系统中的表达

通过对虎源流感病毒A/ Tiger/ Harbin/01/ 2003 (H5N1)的HA 基因进行克隆与序列测定,证明该基因全长为1 731 bp,读码框由1 707个碱基组成,编码568 个氨基酸。对HA 基因的进化分析表明,该基因与H5 亚型流感病毒的HA 基因同源性最高,其HA 裂解位点由6 个碱性氨基酸插入序列(RRRKKR)组成,符合高致病性禽流感病毒的分子特征。将HA 基因克隆入杆状病毒转座载体质粒pFastBacⅠ,构建重组质粒pFastBac-HA;再将该重组质粒转化DH10 Bac 感受态细菌,在体内进行重组,并经三重抗性与蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HA;将Bacmid-HA 转染sf9 细胞,获得重组杆状病毒。经Western-blotting 检测,HA 蛋白在重组杆状病毒中获得表达。用感染重组病毒的sf9 细胞免疫小鼠,2 次免疫后2 周可诱导小鼠产生1∶ 8 ～1∶ 16 的血凝抑制抗体,表明虎源流感病毒的HA 基因在重组杆状病毒系统中得到了正确表达。     

Latent infection of a new alphanodavirus in an insect cell line

Insect BTI-TN-5B1-4 (Tn5) cells have been used extensively with recombinant baculoviruses to express foreign genes. When a recombinant baculovirus containing the hepatitis E virus capsid protein gene was used to infect Tn5 cells, unknown virus particles in addition to the anticipated hepatitis E virus-like particles were produced in the infected cells. The unknown virus particles were 35 nm in diameter and contained RNA that was highly homologous to full-length RNA1 (3,107 bp) and RNA2 (1,383 bp) genomic RNAs of flock house virus. Surprisingly, both RNAs seen in these induced nodavirus particles could be amplified from commercially available Tn5 cells without infection with or induction by a baculovirus. The nucleotide sequences from the purified nodavirus particles and the normal Tn5 cells were identical, demonstrating that the Tn5 cells themselves were latently infected with a nodavirus. However, the generation of nodavirus particles was significantly stimulated by infection with recombinant baculoviruses. Phylogenetic analysis suggested that this new nodavirus belongs to the genus Alphanodavirus in the family Nodaviridae.     

Expression of a biologically active ovine trophoblastic interferon using a baculovirus expression system.

Ovine trophoblast protein (oTP) an embryonic interferon, which plays a key role in maternal recognition of pregnancy, has been expressed in insect cells using a baculovirus expression system. A cDNA coding for oTP was inserted downstream of the strong polyhedrin promoter. Cells infected with recombinant virus produced biologically active oTP and greater than 90% was secreted into the culture medium during infection. High amount of antiviral activity were produced (up to 5 x 10(5) IU per ml of culture medium). Recombinant oTP (roTP) was purified by immunoaffinity chromatography and found to be identical to authentic oTP with respect to molecular mass and N-terminal amino acid sequence.     

HIV—1核蛋白p24在昆虫细胞中的表达

将完整的ＨＩＶ－１ ｐ２４基因克隆到杆状病毒转移质粒中,使用重组转移质粒与野生型杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经筛选获得带有编码ｐ２４基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中表达了ＨＩＶ核蛋白ｐ２４。其重组蛋白的分子量为２４ｋＤ。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人ＨＩＶ－１阳性血清和单克隆抗体所识别。     

Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3

Using the virus vector derived from a baculovirus of Bombyx mori (Bm), we constructed an infectious recombinant virus carrying the mouse interleukin-3 (IL-3) cDNA placed downstream from the polyhedrin promoter. Silkworms infected in vivo with recombinant virus or the silkworm-derived BmN cell line infected in vitro secreted large amounts of IL-3 into hemolymph or culture medium, respectively. On a per volume basis, about 20-fold more activity was found in the culture supernatants of the infected BmN cells and 10000-fold more activity was detected in the hemolymph as compared to supernatants obtained from COS7 monkey cells transfected with plasmid pcD-IL3 using the SV40 early promoter [Yokota et al., Proc. Natl. Acad. Sci. USA 81 (1984) 1070-1074]. Three distinct species of Il-3 of molecular masses, 18, 20 and 22 kDa were produced and all were converted to a 15-kDa protein by N-glycanase digestion, indicating that silkworm cells glycosylated IL-3. The N-terminal amino acid sequences of the IL-3 purified from tissue culture medium and hemolymph were identical to that of mammalian-derived IL-3, showing that silkworm cells recognized the mammalian signal sequence and cleaved it at the correct position. The purified silkworm-produced IL-3 had biological activities indistinguishable from IL-3 produced by mammalian cells as assessed by mast-cell proliferation assays, colony-formation assays using mouse bone marrow cells, and by receptor-binding assays using [125I]IL-3.     

Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region

The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.     

HCV核心蛋白在昆虫细胞中的表达及其免疫学特性的初步评价

通过逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）从丙肝患者的血清中分离出编码完整ＨＣＶ核心蛋白（Ｃ区）的ｃＤＮＡ片段,并将其克隆到杆状病毒转移质粒中。重组转移质粒ＤＮＡ与线性的杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经蚀斑筛选获得了带编码全部核心蛋白基因的重组杆状病毒。重组病毒感染细胞后表达ＨＣＶ核心蛋白,其分子量的为２０ｋＤ。免疫印染和酶联免疫实验表明,此重组蛋白能被人ＨＣＶ阳性血清所识别。动物实验表明此重组蛋白能诱导小鼠产生特异性抗体。     

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.     

Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera.

The terminal proteins TP1 and TP2 are putative products of Epstein-Barr virus (EBV) genes expressed during the latent cycle of the virus. They are predicted to code for 53- and 40-kilodalton integral membrane proteins. We used the baculovirus Autographa californica nuclear polyhedrosis virus as an expression vector to produce TP1 in large amounts in insect cells. The DNA sequences used to express TP1 originated from a TP1 cDNA derived from an M-ABA/CBL1 cDNA library. Rabbit antisera raised against procaryotic TP1 fusion proteins recognized a monomer and a dimer of the recombinant TP1 protein in the infected insect cells. Immunofluorescence studies of living insect cells showed that the recombinant protein is located in the plasma membrane. The insect cells infected with the recombinant baculovirus producing TP1 provided a test system to screen human antisera for TP1 antibodies. A total of 168 human EBV-positive and EBV-negative antisera were studied. TP1 antibodies were detected only in sera from nasopharyngeal carcinoma patients (16 out of 42). Rabbit antiserum raised against the recombinant TP1 protein expressed in the baculovirus system specifically recognized a protein of about 54 kilodaltons in the lymphoblastoid cell lines M-ABA and M-ABA/CBL1 and in the Burkitt's lymphoma cell lines BL18 and BL72. This protein could be located in the total membrane fraction of M-ABA cells and is upregulated by treating the cells with 12-O-tetradecanoylphorbol-13-acetate.     

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.     

Molecular cloning of cDNA for the import precursor of human coupling factor 6 of H(+)-ATP synthase in mitochondria

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat factor 6 as a probe. The sequence was composed of 466 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions on the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame were found to consist of 108 and 76 amino acid residues with molecular weights of 12,596 and 8,969, respectively. The presequence of 32 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells.     

Cloning and expression of a cDNA for the human homolog of mouse T cell and mast cell growth factor P40

A cDNA encoding the human homolog of mouse T-cell and mast cell growth factor P40 was derived from peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin and phorbol myristate acetate. Sequence analysis of the cDNA predicted a precursor protein of 144 amino acids including a signal peptide of 18 residues, a structure identical with that of mouse P40. The homology between the mouse and human proteins is 55% with a perfect conservation of the 10 cysteine residues present in the mature polypeptide. Expression of the cDNA for human P40 in a baculovirus vector yielded a protein capable of enhancing in vitro survival of human T cell lines.     

Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.     

cDNA cloning and sequencing for the import precursor of coupling factor 6 in H(+)-ATP synthase from rat liver mitochondria

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector

A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes.