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PubMed1994

Hepatitis B virus proteins expressed by recombinant vaccinia viruses: influence of preS2 sequence on expression surface and nucleocapsid proteins in human diploid cells.

Fifteen vaccinia virus (VV) recombinants derived from VV strains Praha, LIVP and DD (i.e. Dryvax Wyeth vaccine-derived) and expressing genes for S, preS2-S or c antigens of hepatitis B virus (HBV) were tested in monkey CV-1 cells and human diploid LEP cells. The production of infectious virus was found to be alike in all the recombinants and parental viruses as well. However, several recombinants produced markedly lesser amounts of S and preS2 antigens in LEP cells than in CV-1 cells. This reduction was independent of the parental virus used. There was, however, a relationship between the production of preS2 in CV-1 cells and the production of S and preS2 antigens in LEP cells; in general, recombinants efficiently inducing preS2 antigen formation in CV-1 cells produced markedly reduced amounts of S and preS2 antigens in LEP cells. Reduction of HBV antigen production in LEP cells was not apparent in recombinants expressing only S or c antigens of HBV, and the production of c antigen by double recombinants was not influenced by simultaneous expression of preS2 and S. The various recombinants also differed in the ratio of S:preS2 antigen formation. This difference seemed to be associated with the length of the untranslated leader sequence preceding preS2 but not with the parental virus or cell type used. The titers of antibodies against S and preS2 antigens induced in mice immunized with different recombinants differed markedly. The differences in the ratio of S:preS2 antigen production in vitro were not reflected in vivo by S:preS2 antibody ratio.

Animals↗

PubMed1995

Depolarization-induced 86Rb+ efflux in CHO cells expressing a recombinant potassium channel.

Cells expressing a recombinant human voltage-activated potassium channel (K-channel), Kv1.5, have been used in a functional assay that measures depolarization-stimulated 86Rb+ efflux as an indicator of K-channel function. Neither untransfected nor vector-transfected cells display measurable 86Rb+ efflux under depolarizing conditions. The depolarization-induced 86Rb+ efflux is blocked by standard K-channel blockers quinine, 4-aminopyridine and 3,4-diaminopyridine, but not by tetraethylammonium, quinidine, glibenclamide, or several peptide toxins. The pharmacological profile of the recombinant system reflects that reported for the channel in its native state. In such a system with no observable endogenous background, analysis of recombinant K-channel subtypes allows rapid assessment of pharmacological agents with isoform selectivity and specificity. Inclusion of compounds of unknown activity in an assay such as this could identify agents capable of modulating specific K-channel isoforms. Development of this high through-put assay system for the study of specific isoforms is a critical step in the identification and development of drugs that affect the desired target tissues with predictable pharmacology and minimal side effects due to nonselective K-channel interaction.

Amino Acid Sequence↗

PubMed1993

Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system.

We cloned and expressed the sequences encoding the structural proteins of the hepatitis C virus in a baculovirus eukaryotic expression system. Four recombinant constructs expressed sufficient hepatitis C virus-specific proteins in insect cell culture to allow analysis of protein cleavage, glycosylation and immunoreactivity. Using immunoblot analysis, we detected a 22-kD protein corresponding to the hepatitis C virus capsid protein cleaved from a larger precursor. Recombinant constructs encoding the presumptive envelope (E1) protein produced products ranging from 30 to 35 kD, whereas constructs encoding the presumptive E2/NS1 protein expressed products ranging in size from 68 to 73 kD. The recombinant envelope proteins were glycosylated, as shown by sensitivity to endoglycosidase F digestion, whereas the capsid was not. We examined the immunoreactivity of these recombinant proteins using sera from 50 patients chronically infected with HCV. Forty-seven of 50 of these sera contained antibodies against the capsid, 14 (28%) also had antibodies against E1 and at least 5 (10%) had antibody against E2/NS1. Forty-seven of 50 sera (94%) were viremic, as determined on hepatitis C virus polymerase chain reaction. The three sera that were hepatitis C virus polymerase chain reaction negative did not have envelope antibodies, whereas all sera that had envelope antibodies were also hepatitis C virus polymerase chain reaction positive. Thus antibodies to baculovirus-expressed hepatitis C virus structural proteins, including E1 and E2/NS1, are found in the presence of viremia.

Baculoviridae↗

PubMed2003

Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase.

Recombinant mammalian proteins expressed in E. coli can be difficult to purify in high yield in a soluble and functional form. Various techniques have been described to prevent proteolysis of expressed proteins and/or their sequestering as insoluble aggregates within inclusion bodies. We report conditions for expressing recombinant proteins from E. coli that significantly enhanced the yield of soluble and functional protein. We demonstrate high-yield recovery of a native, high-molecular-weight RNA binding protein without the aid of fusion protein sequence. The principle factor that increased protein yield was the induction of protein expression in a late log phase culture, although reduced temperature during the induction and a low IPTG concentration also contributed to a higher yield.

Cell Culture Techniques↗

PubMed2002

Site-specific integration of the double-mutation glucose isomerase (GIG138PG247D) gene in Streptomyces lividans and its stable expression.

A recombinant expression plasmid pYH12, containing the double-mutation glucose isomerase (GIG138PG247D, GI2) coding gene and its natural regulatory sequence, was constructed for site-specific integration in Streptomyces. The resulting plasmid was introduced into Streptomyces lividans TK54 by protoplast transformation and two apramycin-resistance (AmR) transformants, designated GY2 and BY7, respectively, were obtained further based on enzyme assays. These results for polymerase chain reaction (PCR), Dot blot, and recovery of cloned fragments from the transformant chromosome indicated that the GI2 gene was integrated into the S. lividans chromosome by site-specific recombination, and which was further verified by Southern blot. We found that the free form of plasmid pYH12 co-existing with the integrated form was present in S. lividans. SDS-PAGE analysis showed that the GI2 gene was expressed in S. lividans. The intracellular GI2 specific activity was 1.15 U/mg. The stability of integrants demonstrated that the cloned GI2 gene was stably integrated and expressed even in the absence of selective pressure.

Aldose-Ketose Isomerases↗

PubMed1989

Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses.

Recombinant baculoviruses containing the coding sequences of the viral structural proteins, i.e., the capsid (C) protein, the precursor to premembrane (preM) protein, and the envelope (E) protein, as well as a nonstructural protein, NS1, of Japanese encephalitis virus (JEV) were constructed. Infection of Spodoptera frugiperda cells with these recombinant viruses produced PreM and E proteins. The E proteins synthesized by the recombinants were shown to be glycosylated and similar in size to the authentic E protein. The E protein was found on the surface of infected cells. The antigenic properties of recombinant E proteins were evaluated using a panel of monoclonal antibodies produced against JEV E protein. It was demonstrated that all of the epitopes detectable on the authentic JEV E protein were present on the recombinant E protein expressed by a recombinant baculovirus containing the coding sequence for a part of C, PreM, E, and a part of NS1 proteins. However, for E protein expressed by a recombinant baculovirus having the coding sequence of only a part of PreM, but all of E and a part of NS1, one of the flavivirus cross-reactive epitopes was not detected. Mice immunized with cells infected with the recombinant baculoviruses developed neutralization antibodies.

Animals↗

PubMed2006

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression.

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.

Escherichia coli↗

PubMed2000

Identification by electron microscopy of the maturation steps in vaccinia virus morphogenesis inhibited by the interferon-induced enzymes, protein kinase (PKR), 2-5A synthetase, and nitric oxide synthase (iNOS).

Interferons (IFN) play a major role as a first-line host defense mechanism against viral infections. As treatment of animal cells with IFN induces a large number of genes, it has been difficult to assign the role of these genes in the antiviral action of IFN. Vaccinia virus (VV) is an ideally suited system to study IFN action because all steps in viral morphogenesis can be followed easily by electron microscopy (EM) of ultrathin sections from infected cells. To define the role of IFN-induced genes in viral morphogenesis, we have independently expressed from VV recombinants in primary chicken embryo fibroblast (CEF) cells each of the three IFN-induced genes encoding protein kinase (PKR), 2-5A synthetase, and inducible nitric oxide synthase (iNOS). By EM analysis, we have identified the steps in VV morphogenesis that are affected by each of the IFN-induced enzymes in comparison with untreated and IFN-treated cells. We found that in cells pretreated with IFN and infected with VV, immature virus (IV) is formed, but further stages of maturation are blocked. In cells infected with a VV recombinant expressing PKR (VV-PKR), there is severe inhibition on virus factories, and only few IV are formed. In cells infected with a VV recombinant expressing 2-5A synthetase (VV-2-5A), VV assembly is inhibited at or after IV formation. In cells infected with a VV recombinant expressing iNOS (VV-iNOS), all stages in VV morphogenesis are observed but with aberrant forms. In addition to the effects on viral assembly, in cells infected with either VV-PKR, VV-2-5AS, or VV-iNOS, there is nucleus condensation characteristic of apoptosis. Our findings have identified the steps in VV morphogenesis inhibited by PKR, 2-5A, and iNOS, provided a distinction between these effects, and highlighted a functional redundancy of the IFN system to block viral infection and to induce apoptosis.

2',5'-Oligoadenylate Synthetase↗

PubMed1997

Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopneumoniae antigen (NrdF).

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a commercially expensive respiratory disease of swine. Salmonella typhimurium SL3261 was used as a live carrier of plasmid pKF1, which encodes a 15-kDa recombinant M. hyopneumoniae protein. This expressed recombinant protein consists of the carboxy-terminal 11 kDa of a 42-kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Rabbit anti-15-kDa serum was able to inhibit the growth of viable M. hyopneumoniae J in vitro. When used as a live oral vaccine, S. typhimurium SL3261(pKF1) induced a significant secretory immunoglobulin A immune response in the lungs of mice orally immunized against the M. hyopneumoniae antigen. Utilization of live oral vaccines expressing potentially protective M. hyopneumoniae proteins, such as the NrdF antigen, which can stimulate a lung mucosal response against surface-accessible proteins may provide a cost-effective alternative to the present control strategies used for porcine enzootic pneumonia.

Administration, Oral↗

PubMed1993

Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals.

Vesicular stomatitis is a viral disease of cattle, pigs, and horses. The disease is characterized by vesicular lesions on the epithelium of the mouth, feet, and teats. The pathological lesions are virtually indistinguishable from that of foot-and-mouth disease. We have developed a recombinant baculovirus that expresses the nucleocapsid (N) protein of the New Jersey serotype of vesicular stomatitis virus (VSVNJ) in insect cells (Sf9) and larvae (Spodoptera exigua). The gene was expressed under control of the polyhedrin promoter as a fusion or nonfusion protein. The recombinant N protein expressed in insect cells could not be distinguished from N protein produced in VSVNJ-infected CHO cells by immunological and biochemical analyses. The level of expression of N as a percentage of the total protein in Sf9 cells was 41% for the fusion and 60% for the nonfusion protein. Higher level (68%) of expression of the nonfusion N protein was obtained in larvae. Recombinant N protein was used in an ELISA to distinguish animals vaccinated with a recombinant VSV glycoprotein from those exposed to the whole virus by infection or classical vaccine. Lysate of a single infected larva (0.2-0.3 g) was adequate for coating ELISA plates to perform 10,000 serum assays in duplicate.

Amino Acid Sequence↗

PubMed2002

Construction, expression and biological assessment of BPI23-Fcgamma1 recombinant protein prokaryotic expression vector.

OBJECTIVE: To construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein. METHODS: Genes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method. RESULTS: (1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization. CONCLUSION: pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.

Antimicrobial Cationic Peptides↗

PubMed1999

Insect cells as hosts for the expression of recombinant glycoproteins.

Baculovirus-mediated expression in insect cells has become well-established for the production of recombinant glycoproteins. Its frequent use arises from the relative ease and speed with which a heterologous protein can be expressed on the laboratory scale and the high chance of obtaining a biologically active protein. In addition to Spodoptera frugiperda Sf9 cells, which are probably the most widely used insect cell line, other mainly lepidopteran cell lines are exploited for protein expression. Recombinant baculovirus is the usual vector for the expression of foreign genes but stable transfection of - especially dipteran - insect cells presents an interesting alternative. Insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. For large scale culture, conditions have been developed which meet the special requirements of insect cells. With regard to protein folding and post-translational processing, insect cells are second only to mammalian cell lines. Evidence is presented that many processing events known in mammalian systems do also occur in insects. In this review, emphasis is laid, however, on protein glycosylation, particularly N-glycosylation, which in insects differs in many respects from that in mammals. For instance, truncated oligosaccharides containing just three or even only two mannose residues and sometimes fucose have been found on expressed proteins. These small structures can be explained by post-synthetic trimming reactions. Indeed, cell lines having a low level of N-acetyl-beta-glucosaminidase, e.g. Estigmene acrea cells, produce N- glycans with non-reducing terminal N-acetylglucosamine residues. The Trichoplusia ni cell line TN-5B1-4 was even found to produce small amounts of galactose terminated N-glycans. However, there appears to be no significant sialylation of N-glycans in insect cells. Insect cells expressed glycoproteins may, though, be alpha1,3-fucosylated on the reducing-terminal GlcNAc residue. This type of fucosylation renders the N-glycans on one hand resistant to hydrolysis with PNGase F and on the other immunogenic. Even in the absence of alpha1,3-fucosylation, the truncated N-glycans of glycoproteins produced in insect cells constitute a barrier to their use as therapeutics. Attempts and strategies to "mammalianise" the N-glycosylation capacity of insect cells are discussed.

Animals↗

PubMed1994

Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus.

The ability to express recombinant genes in the coronary vasculature and the myocardium holds promise for the treatment of a number of acquired and inherited cardiovascular diseases. Previous in vivo gene transfer approaches in the heart have been limited by relatively low efficiencies of gene transduction. In this report, we demonstrate that catheter-mediated infusion of replication-defective adenovirus into the coronary arterial circulation in vivo represents a novel and efficient method for the induction of recombinant gene expression in both the coronary arteries and the myocardium. A single intracoronary infusion of 2 x 10(9) - 1 x 10(10) p.f.u. of adenovirus resulted in high level recombinant gene expression in both the coronary arteries and surrounding myocardium of adult rabbits for at least 2 weeks. No inflammatory response or myocardial necrosis was observed following the adenovirus infusions. The polymerase chain reaction (PCR) was used to assess the tissue distribution of infection following intracoronary infusion of adenovirus. Adenovirus DNA was detected by PCR in the livers, kidneys, lungs, brains and testes of animals 5 days after virus infusion. Percutaneous transluminal gene transfer (PTGT) into the heart by intracoronary infusion of replication-defective adenovirus represents a relatively non-invasive and efficient method of inducing recombinant gene expression both in the coronary arterial wall and in the surrounding myocardium.

Adenoviridae↗

PubMed2004

Down-regulation of CXCR4 expression in MT4 cells by a recombinant vector expressing antisense RNA to CXCR4 and its potential anti-HIV-1 effect.

CXC-chemokine receptor (CXCR4) is one principle co-receptor for the entry of T cell line (T)-tropic HIV-1 virus into a cell. In order to find more efficacious therapeutic possibilities for people with an HIV-1 infection, we explored the inhibitory effects of antisense RNA on CXCR4 expression in MT4 cells. First, we used to RT-PCR to obtain DNA fragments from healthy adult peripheral blood mononuclear cells; these fragments targeted the initiation region of CXCR4 mRNA translation. We then constructed a recombinant retroviral vector, pLXSN-X4a (containing antisense RNA to CXCR4). After packaging by PA317 cells, the pseudovirion of the recombinant vector had formed and succeeded in transfecting MT4 cells (a kind of T-tropic HIV-1 susceptibility cell line). The PCR and RT-PCR results showed that the recombinant vector had integrated into the genome of MT4 cells and had been transcribed. The expression of CXCR4 on the surface of MT4 cells transfected with antisense RNA was reduced by 30%, compared with those cells transfected with blank vector or untransfected cells. No change in the DNA synthesis rates or in cell proliferation was found in any of the transfected cells. After a challenge with HIV-1 SF33, the cells transfected with antisense RNA vector (pLXSN-X4a) produced reduced p24 levels compared with the cells transfected with blank vector (pLXSN) or untransfected cells. These results indicated that these CXCR4-antisense expressing cells could resist T-tropic HIV-1 infection and could retain normal biological functions. These studies provide useful data for further experiments in this area.

Cell Line, Transformed↗

PubMed2001

Analysis of Borrelia burgdorferi vlsE gene expression and recombination in the tick vector.

Expression and recombination of the antigenic variation vlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assess vlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34 degrees C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in the B. burgdorferi natural history. To assess vlsE recombination, nymphs were capillary fed the B. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. The vlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in the vlsE sequence. In contrast, vlsE cassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsE recombination does not occur in the tick vector.

Animals↗

PubMed2005

[Construction and expression of recombinant adenovirus containing human thymic stromal lymphopoietin gene].

AIM: To construct and express recombinant adenovirus bearing human thymic stromal lymphopoietin (TSLP) gene. METHODS: TSLP gene amplified from human fetal lung cells was first cloned into eukaryotic expression vector pcDNA3.1, and then subcloned into shuttle vector pShuttle. The resultant plasmid was subsequently cotransformed into E. coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid containing TSLP was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. The TSLP gene of the recombinant virus was detected by PCR, and its expression was analyzed by Western blot. RESULTS: Recombinant adenovirus vector bearing human TSLP gene was constructed by homologous recombination in E.coli, and recombinant adenovirus was obtained by transfecting HEK293 cells with this infectious DNA. PCR test indicated that TSLP gene was successfully integrated into the adenoviral genome, and the titer of the recombinant Ad reached 1 x 10(11) pfu/L. Meanwhile, expression of TSLP in the infected Hela cells was confirmed by Western blot. CONCLUSION: The construction of recombinant adenovirus bearing human TSLP gene and its expression mediated by this adenovirus pave a foundation for the study on the biological function of this novel cytokine.

Adenoviridae↗

PubMed1993

Induction of neutralizing antibodies by varicella-zoster virus gpII glycoprotein expressed from recombinant vaccinia virus.

The gpII glycoprotein of varicella-zoster virus (VZV) was produced in CV1 cells via vaccinia virus recombinants. Two different DNA constructs were expressed: the first one encodes the complete gpII protein (gpII s+a+) and the second a truncated species lacking the membrane anchorage domain (gpII s+a-). To achieve expression both coding sequences had to be engineered at the 5' end by substituting the unusually short (24 bp) natural signal sequence by a more conventional one encoding 29 amino acids. Recombinant gpII proteins were detected in vaccinia virus-infected cells by ELISA and immunoprecipitation. Both forms of recombinant gpII proteins were produced as glycosylated single-chain molecules of respectively 110K and 90K. Upon reduction these were only partially converted into subunits. A rabbit infected with the vaccinia virus recombinant expressing the complete gpII produced antibodies which recognized VZV antigens and neutralized VZV infectivity in vitro, independent of complement.

Amino Acid Sequence↗

PubMed2003

[Construction of recombinant eukaryotic expression vector of NKG2D gene and its expression in COS-7 cells].

OBJECTIVE: To construct recombinant eukaryotic expression vector of NKG2D, and to examine its expression in COS-7 cells. METHODS: Human NKG2D cDNA was obtained from peripheral blood mononuclear cells using RT-PCR, and then the target gene was cloned into pMD18-T vector. A eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique, and was confirmed by double restriction enzymes digestion and DNA sequence analysis. The recombinant plasmid with encapsuled lipofectamine was transfected into COS-7 cells, and the transfected COS- The 7 cells containing expressive target gene were confirmed by RT-PCR and flow cytometry. RESULTS: The sequence of NKG2D cDNA obtained from the recombinant eukaryotic expression vector was identical with that published on GeneBank. The NKG2D gene was expressed successfully in COS-7 cells. CONCLUSION: The recombinant expression plasmid containing NKG2D gene is constructed successfully. After being transfected to COS-7 cells, the NKG2D protein is expressed by the engineering COS-7 cells line, which lays a foundation for further studying biological activity of NKG2D.

Animals↗