جستجوی مقالات مرتبط با کلیدواژه « هماگلوتینین » در نشریات گروه « پزشکی »

Influenza virus, which is a decisive agent of influenza, attracted interest because of the event of the annual epidemic among a wide range of animal and human hosts. This study aimed to express the hemagglutinin protein of Iranian swine Influenza A (H1N1) through a baculovirus system in SF9 cells to produce a new recombinant vaccine.

Hemagglutinin gene of Swine H1N1 virus was amplified with specific primers containing restriction enzymes site, and then cloned. Afterward, the vector was transformed to DH10Bac using Bac-to-Bac system in order to produce a recombinant bacmid. Hemagglutinin expression and its biological activity were assessed using molecular and immunization tests.

The target rHA in length, 1710 bp, was produced and expressed in transfected SF9 cells with a size of ~66 kDa. The infected cells expanded in size and their nucleus, and desiccated from the surface of the cell culture as a granular. They could absorb chick red blood cells (RBCs), and appear as cell aggregates forty-eight postinfection. The fact that infected cells were unable to form cell clamp showed the test's accuracy and inhibition of hemodesorption activity. The amount of protein obtained was 10.76 µg/100 µl, equal to 0.1 mg/ml.

The baculovirus expression system could express the recombinant protein in the insect cell. Therefore, it may be a well-suited alternative to produce a new generation of the vaccine instead of egg-based and cell-culturebased generation vaccines.

According to marked advances in bioinformatics studies, development of influenza vaccines has been greatly modified in many studies. In this study, we have designed a multi-epitope recombinant protein, consisting of several conserved epitopes from Hemagglutinin protein of the H1N1 and H5N1 strains of Influenza virus by immunoinformatics approaches.

The registered sequences of hemagglutinin proteins from H1N1 and H5N1 strains have been analyzed and selected epitopes have been chosen and directed for further analysis. After investigation of immunological and physicochemical parameters, the selected regions were fused together by linkers and sequence of target constructs was determined following codon optimization. The target construct has been integrated into BamHI/XhoI cleavage sites of PET32a+ expression vector.

A physicochemical analysis revealed that the designed recombinant protein has 214 amino acid with molecular weight of 22.451 kDa. The isoelectric point was determined as 7.25 , solubility of 100% and  Extinction coefficient of 60170 M-1cm-1.finally, after codon optimization, the Codon Adaptation Index (CAI) was calculated as 0.95.

According to these results, it has been predicted that designed multiepitop recombinant protein has reasonable and unique immunological properties. The expressed protein could be used for future immunological studies against type A influenza virus

 The H1N1 influenza virus is a highly pathogenic virus that threatens human life. Vaccination is an effective way of preventing and controlling influenza. Production of recombinant hemagglutinin in the baculovirus expression system, in the insect eukaryotic cell substrate (Sf9), has been suggested as an effective strategy.

The H1N1 influenza virus hemagglutinin gene sequence was prepared from National Center for Biotechnology Information (NCBI). After designing a specific primer, the sequence was provided using restriction digestion, cloned into pFastBacHTA plasmid, and transferred to the DH10Bac cell to produce a recombinant bacmid. After extracting the relevant plasmid, it was transfused into the insect cell; and after the expression of the protein by Sf9 cell, the presence of recombinant protein was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot methods.

The hemagglutinin gene (654 bp) was cloned in pFastBacHTA plasmid using the two enzymes of BamHI and Xhol. Sf9 cell expressed a protein weighing approximately 60 kDa after receiving the recombinant bacmid protein. The extracted protein was identified and confirmed using SDS-PAGE and Western blot methods; and protein concentration was measured by Lowry method.

 The baculovirus system is useful for the production of proteins with complex structures. Generally, it can be concluded that this protein is highly expressed in insect cells. Due to the similarity of this system with the human system, it can be a suitable alternative for embryonic eggs in the future, and can be used in vaccination.

Each year, Human influenza A (H1N1) virus causes moderate to severe infections with a high prevalence throughout the world. Accordingly, the rapid, sensitive and cost-effective laboratory diagnosis based on viral antigen detection is important. Moreover, the generation of specific antibodies directed against Influenza antigens is essential to the success of both basic and applied research programs. Hemagglutinin (HA) is the major surface envelope glycoprotein of influenza virus, which is subsequently cleaved into two subunits, HA1 and HA2. Since most antigenic sites are in the HA1 domain of HA, HA1 domain of influenza virus was studied as antigen to produce polyclonal antibody.
In this experimental study we expressed and purified the recombinant HA1 protein in the second half of 2015 at department of influenza and other respiratory viruses, Pasteur Institute of Iran and then prepared the polyclonal rabbit antibody against it. The vector of pET28aHA1 expressing HA1-His tagged protein of H1N1 influenza A/PR/8/34 virus was used for large scale production of HA1 into E. Coli (BL21). By changing expression conditions such as IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentration, time and temperature of incubation, the expression conditions for HA1 were optimized. The total cell protein harvested and purified by nickel affinity chromatography. All above mentioned experiments monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The efficiency of HA1 recombinant protein was high, equal to 400-600 mg/ml of cell lysate. The polyclonal antibody was prepared by immunizing the rabbits using recombinant HA1 with Freund’s adjuvant according to standard protocols. Efficiency of the antiserum evaluated by enzyme linked immunosorbent assay (ELISA). Determination of antibody level in the collected antiserum using serum-based ELISA showed that the specific antibody has risen well through the immunization schedule.
Our data shows that this polyclonal antibody has potential to be produced in rabbit. It will also be used in the future in influenza diagnosis as well as in other immunological applications such as western blot analyses, immunocytochemistry, and immunohistochemistry.

Influenza viruses cause acute respiratory infections in humans, which can often be accompanied by pandemics with varying rates of illness and even a fetal end. Current vaccines target the variable globular part of hemagglutinin (HA); so, their effectiveness is limited and they need perpetual updating. To prepare universal vaccine, scientist's effort is focused on conserved antigenic parts of influenza virus such as hemagglutinin stalk domain which is more conserved compare to its globular head. The aim of this study was evaluating the immunogenicity of influenza A (H1N1) HA2 peptide in mouse model. To prepare HA2 protein, the expression vector encoding the gene of interest (PET-28a/HA2), was transformed into Escherichia coli BL21-DE3 competent cells. Recombinant HA2 protein was extracted and purified using Ni-TED columns under denaturing conditions, refolded and desalted via step-wise dialysis. The HA2 concentration was determined via using Bradford protein assay. Six-week-old BALB/c mice in different groups were immunized intradermally with HA2 alone or supplemented with Freund or Lm.HSP70221-604 as adjuvants. Two groups were injected with HSP70 and phosphate buffered saline (PBS) as negative controls. Humeral immunity in vaccinated mice was measured using the enzyme-linked immunosorbent assay (ELISA); and finally, mice were challenged with one lethal dose (LD90) of PR8 virus. The protein expression, extraction and purification stages were confirmed using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis. The highest antibody response was measured in mice vaccinated with HA2-Freund, as expected. Specific antibody response in group received HA2-HSP70 was of higher titer than HA2 alone. More over HA2-HSP70 group surprisingly showed higher level of protection against lethal H1N1 (PR8) challenge compared to other groups. It has been proved that HSP70 can induce specific response and innate immunity via stimulating CD8+ T-Cells, CD40, TLR2, TLR4 and cytokine secretion. Our results showed that HA2 alone or supplemented with HSP70 protected mice against lethal influenza challenge and could be considered as a universal vaccine.

The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2) for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2.

This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete) and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID) in both forms, Single RID (SRID) and Double RID (DRID). Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay.

Sedimentary line and zone was observed in RID assays (SRID and DRID) represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites.

These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.

Influenza virus is a globally important respiratory pathogen causing high degree of morbidity and mortality annually. The novel Influenza virus (A/H1N1) which involved many populations of the world in 2009 is a sort of triple reassortment between swine، bird and human viruses. Given the important role of hemagglutinin in the infectivity of influenza virus، genome sequencing of this protein and investigation of its changes seems necessary. In this experimental study، the viral genome was extracted from clinical throat swab samples، in which the presence of swine influenza genome has been confirmed by Real-time PCR according to WHO protocol in Influenza Research Lab، Pasteur Institute of Iran. Full-length of HA genome was amplified using specific primers by one step-RT-PCR، cloned into pGEM-T Easy vector followed by identification with restriction enzyme analysis and sequencing. Full genome of novel influenza A/H1N1 from clinical samples was amplified by PCR and the expected 1777 bp segment PCR product was visualized by electrophoresis، gel purified، cloned into pGEM-TEasy vector and then sequenced. Analysis of sequencing was accomplished by chromas software (version 1. 45-Australia) and the nucleotide sequence data was deposited in GenBank database under the accession number: “HQ419001. 1”. The result of sequencing was well-matched with the recommended vaccine strain and other registered sequences in NCBI.

Influenza virus A (H1N1) is an important subtype of the influenza respiratory viruses, which has important worldwide implications. Hemagglutinin (HA), an important viral antigen, is responsible for binding to human cell receptors leading to an onset of the disease process. Considering the critical role of viral attachment, this study focuses on the extraction and cloning of HA and its large subunit HA1 genes to generate recombinant baculovirus shuttle vectors (bacmid) in order to produce recombinant proteins in insect cells. Human influenza virus A/New Caledonia 99/20/(H1N1) was propagated in MDCK cell culture. Total viral RNA was extracted using easy-red solution. The full-length HA genome and HA1 fragment were amplified by RT- PCR using specific primers, cloned into a pGEM®-TEasy vector, and then subcloned into a pFastBac HT plasmid. Finally, recombinant bacmids that contained the genes of interest were produced in E. coli DH10Bac™ cells. Expected PCR products of HA genes were evaluated through gel electrophoresis and restriction enzyme analysis. Recombinant pGEM®-TEasy vectors and pFastBac HT donor plasmids were confirmed by PCR, digestion, and sequencing. Construction of recombinant bacmid DNA was verified by using blue-white colony screening, overnight electrophoresis, and PCR analysis that used either pUC/M13 or gene-specific primers. In this study, we have successfully constructed recombinant Bacmid DNA that encoded the full-length HA genome and its HA1 subunit. We intend to transfect sf9 insect cells with these constructs to generate recombinant baculovirus and produce large amounts of desired proteins for future studies.

Influenza is a contagious respiratory infectious disease out breaking in cold seasons of the year. The outbreak of the new influenza A (H1N1) virus in 2009 involved large populations of the world with considerable mortality. Hemagglutinin (HA) molecule, the main surface glycoprotein of the influenza virus, is one of the key factors for serological diagnostic kits and vaccine development. Thus establishment of HA gene bank of the circulating influenza viruses is essential in gaining quick access to large amounts of protein. The first step in providing such a bank is detection and isolation of HA full genome and its subunits by using specific primers and cloning them in proper vectors. For this purpose, using standard virus genome (A/New Caledonia/20/99(H1N1)) cultured on MDCK cell, HA coding gene was proliferated by RT-PCR using specific primers. Isolation and cloning of the HA gene was verified by RT-PCR, enzyme digestion and determining nucleotide synonymy. Through the use of specific cloning primers, different HA gene constructs were propagated for expression of the gene in insect cells and E.coli bacteria. The results indicated the complete compatibility of the extracted HA gene with the influenza (A/New Caledonia/20/99(H1N1)) hemagglutinin. It makes it possible to use the gene as a source of cloning in a variety of eukaryotic and prokaryotic expression systems