Cloning and Expression of Truncated Spike (Sf200) Glycoprotein of Infectious Bronchitis Virus (IBV) in Escherichia coli, and its Immunogenicity to Mice
Complete S1 gene of the Infectious Bronchitis Virus (IBV) was amplified and cloned into transfer vector. Truncated S1 gene designated as Sf200 (containing five antigenic sites located at 24-61, 291-398 and 497-543 amino acid residues of S1 glycoprotein) were amplified by overlap PCR, cloned into pro...
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| Veröffentlicht in: | Pakistan journal of zoology 2018-06, Vol.50 (3) |
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| container_title | Pakistan journal of zoology |
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| creator | Zeshan, Basit Saleem, Mushtaq A. Wattoo, Javed Iqbal Arshad, Mohd Mokhtar Mohamed, Maizan |
| description | Complete S1 gene of the Infectious Bronchitis Virus (IBV) was amplified and cloned into transfer vector. Truncated S1 gene designated as Sf200 (containing five antigenic sites located at 24-61, 291-398 and 497-543 amino acid residues of S1 glycoprotein) were amplified by overlap PCR, cloned into prokaryotic expression vector resulting pET-Sf200 and confirmed the construct by sequencing. The recombinant plasmid was identified by restriction enzyme and sequencing analysis. The in vitro expression of the truncated protein was analyzed in E. coli with a molecular weight of 38kDa determined through SDS-PAGE and confirmed by Western blotting. The recombinant truncated protein was then purified from the culture media. The immunogenicity of the protein was studied in an animal experiment on mice, in which mice were injected subcutaneously. These findings suggest that the truncated Sf200 expressed in the pET-32a (+) prokaryotic vector can be used as antigen to detect antibodies against IBV. |
| doi_str_mv | 10.17582/journal.pjz/2018.50.3.989.994 |
| format | Article |
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Truncated S1 gene designated as Sf200 (containing five antigenic sites located at 24-61, 291-398 and 497-543 amino acid residues of S1 glycoprotein) were amplified by overlap PCR, cloned into prokaryotic expression vector resulting pET-Sf200 and confirmed the construct by sequencing. The recombinant plasmid was identified by restriction enzyme and sequencing analysis. The in vitro expression of the truncated protein was analyzed in E. coli with a molecular weight of 38kDa determined through SDS-PAGE and confirmed by Western blotting. The recombinant truncated protein was then purified from the culture media. The immunogenicity of the protein was studied in an animal experiment on mice, in which mice were injected subcutaneously. These findings suggest that the truncated Sf200 expressed in the pET-32a (+) prokaryotic vector can be used as antigen to detect antibodies against IBV.</description><identifier>ISSN: 0030-9923</identifier><identifier>DOI: 10.17582/journal.pjz/2018.50.3.989.994</identifier><language>eng</language><publisher>Lahore: AsiaNet Pakistan (Pvt) Ltd</publisher><subject>Adenoviruses ; Amino acids ; Amplification ; Animal vaccines ; Antibodies ; Antigens ; Bronchitis ; Cloning ; Culture media ; Disease ; E coli ; Eggs ; Embryos ; Escherichia coli ; Gel electrophoresis ; Glycoproteins ; Immunogenicity ; Infectious diseases ; Mice ; Molecular chains ; Molecular weight ; Polyclonal antibodies ; Polypeptides ; Positron emission ; Proteins ; S1 gene ; Sodium lauryl sulfate ; Tomography ; Viruses ; Western blotting</subject><ispartof>Pakistan journal of zoology, 2018-06, Vol.50 (3)</ispartof><rights>Copyright 2018 Pakistan Journal of Zoology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids></links><search><creatorcontrib>Zeshan, Basit</creatorcontrib><creatorcontrib>Saleem, Mushtaq A.</creatorcontrib><creatorcontrib>Wattoo, Javed Iqbal</creatorcontrib><creatorcontrib>Arshad, Mohd Mokhtar</creatorcontrib><creatorcontrib>Mohamed, Maizan</creatorcontrib><title>Cloning and Expression of Truncated Spike (Sf200) Glycoprotein of Infectious Bronchitis Virus (IBV) in Escherichia coli, and its Immunogenicity to Mice</title><title>Pakistan journal of zoology</title><description>Complete S1 gene of the Infectious Bronchitis Virus (IBV) was amplified and cloned into transfer vector. Truncated S1 gene designated as Sf200 (containing five antigenic sites located at 24-61, 291-398 and 497-543 amino acid residues of S1 glycoprotein) were amplified by overlap PCR, cloned into prokaryotic expression vector resulting pET-Sf200 and confirmed the construct by sequencing. The recombinant plasmid was identified by restriction enzyme and sequencing analysis. The in vitro expression of the truncated protein was analyzed in E. coli with a molecular weight of 38kDa determined through SDS-PAGE and confirmed by Western blotting. The recombinant truncated protein was then purified from the culture media. The immunogenicity of the protein was studied in an animal experiment on mice, in which mice were injected subcutaneously. These findings suggest that the truncated Sf200 expressed in the pET-32a (+) prokaryotic vector can be used as antigen to detect antibodies against IBV.</description><subject>Adenoviruses</subject><subject>Amino acids</subject><subject>Amplification</subject><subject>Animal vaccines</subject><subject>Antibodies</subject><subject>Antigens</subject><subject>Bronchitis</subject><subject>Cloning</subject><subject>Culture media</subject><subject>Disease</subject><subject>E coli</subject><subject>Eggs</subject><subject>Embryos</subject><subject>Escherichia coli</subject><subject>Gel electrophoresis</subject><subject>Glycoproteins</subject><subject>Immunogenicity</subject><subject>Infectious diseases</subject><subject>Mice</subject><subject>Molecular chains</subject><subject>Molecular weight</subject><subject>Polyclonal antibodies</subject><subject>Polypeptides</subject><subject>Positron emission</subject><subject>Proteins</subject><subject>S1 gene</subject><subject>Sodium lauryl sulfate</subject><subject>Tomography</subject><subject>Viruses</subject><subject>Western blotting</subject><issn>0030-9923</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>BENPR</sourceid><recordid>eNo1kc9KAzEQxvegYKl9h4AgLdh1Ntm_F8GWWguKB7XXkE2TNnWbrEkWrC_i6xrbOpeBmW9m-OYXRdcJxEmRlfh2azqrWRO32-9bDEkZZxCTuCqruKrSs6gHQGBcVZhcRAPnthAizTOMy170M22MVnqNmF6h2VdrhXPKaGQkerOd5syLFXpt1YdAw1eJAUZo3uy5aa3xQh10Cy0F98p0Dk2s0XyjvHJoqWwoDBeT5QgF3czxjbAqNBniplE3h4PKO7TY7Tpt1kIrrvweeYOeFReX0blkjRODU-5H7w-zt-nj-OllvpjeP405rlI_riWQHBdJlmCckVVas5pBUZZpnQjCS5CS55CwPBNQpyzNc5YG61jKohYshxXpR1fHvcHQZyecp6dnOoqhgCIL7yyC6u6o4tY4Z4WkrVU7Zvc0AXpg8D9GAwP6x4BmQAkNDGhgQH4BI5-Bdw</recordid><startdate>20180601</startdate><enddate>20180601</enddate><creator>Zeshan, Basit</creator><creator>Saleem, Mushtaq A.</creator><creator>Wattoo, Javed Iqbal</creator><creator>Arshad, Mohd Mokhtar</creator><creator>Mohamed, Maizan</creator><general>AsiaNet Pakistan (Pvt) Ltd</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7SS</scope><scope>7XB</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>LK8</scope><scope>M2P</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PYCSY</scope><scope>Q9U</scope><scope>RC3</scope></search><sort><creationdate>20180601</creationdate><title>Cloning and Expression of Truncated Spike (Sf200) Glycoprotein of Infectious Bronchitis Virus (IBV) in Escherichia coli, and its Immunogenicity to Mice</title><author>Zeshan, Basit ; Saleem, Mushtaq A. ; Wattoo, Javed Iqbal ; Arshad, Mohd Mokhtar ; Mohamed, Maizan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c294t-bf036271512253d4baba07884b1e3c80ffc601a65e0b4a466a40462ff7bea60d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Adenoviruses</topic><topic>Amino acids</topic><topic>Amplification</topic><topic>Animal vaccines</topic><topic>Antibodies</topic><topic>Antigens</topic><topic>Bronchitis</topic><topic>Cloning</topic><topic>Culture media</topic><topic>Disease</topic><topic>E coli</topic><topic>Eggs</topic><topic>Embryos</topic><topic>Escherichia coli</topic><topic>Gel electrophoresis</topic><topic>Glycoproteins</topic><topic>Immunogenicity</topic><topic>Infectious diseases</topic><topic>Mice</topic><topic>Molecular chains</topic><topic>Molecular weight</topic><topic>Polyclonal antibodies</topic><topic>Polypeptides</topic><topic>Positron emission</topic><topic>Proteins</topic><topic>S1 gene</topic><topic>Sodium lauryl sulfate</topic><topic>Tomography</topic><topic>Viruses</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zeshan, Basit</creatorcontrib><creatorcontrib>Saleem, Mushtaq A.</creatorcontrib><creatorcontrib>Wattoo, Javed Iqbal</creatorcontrib><creatorcontrib>Arshad, Mohd Mokhtar</creatorcontrib><creatorcontrib>Mohamed, Maizan</creatorcontrib><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Environmental Science Collection</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><jtitle>Pakistan journal of zoology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zeshan, Basit</au><au>Saleem, Mushtaq A.</au><au>Wattoo, Javed Iqbal</au><au>Arshad, Mohd Mokhtar</au><au>Mohamed, Maizan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning and Expression of Truncated Spike (Sf200) Glycoprotein of Infectious Bronchitis Virus (IBV) in Escherichia coli, and its Immunogenicity to Mice</atitle><jtitle>Pakistan journal of zoology</jtitle><date>2018-06-01</date><risdate>2018</risdate><volume>50</volume><issue>3</issue><issn>0030-9923</issn><abstract>Complete S1 gene of the Infectious Bronchitis Virus (IBV) was amplified and cloned into transfer vector. Truncated S1 gene designated as Sf200 (containing five antigenic sites located at 24-61, 291-398 and 497-543 amino acid residues of S1 glycoprotein) were amplified by overlap PCR, cloned into prokaryotic expression vector resulting pET-Sf200 and confirmed the construct by sequencing. The recombinant plasmid was identified by restriction enzyme and sequencing analysis. The in vitro expression of the truncated protein was analyzed in E. coli with a molecular weight of 38kDa determined through SDS-PAGE and confirmed by Western blotting. The recombinant truncated protein was then purified from the culture media. The immunogenicity of the protein was studied in an animal experiment on mice, in which mice were injected subcutaneously. These findings suggest that the truncated Sf200 expressed in the pET-32a (+) prokaryotic vector can be used as antigen to detect antibodies against IBV.</abstract><cop>Lahore</cop><pub>AsiaNet Pakistan (Pvt) Ltd</pub><doi>10.17582/journal.pjz/2018.50.3.989.994</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Adenoviruses Amino acids Amplification Animal vaccines Antibodies Antigens Bronchitis Cloning Culture media Disease E coli Eggs Embryos Escherichia coli Gel electrophoresis Glycoproteins Immunogenicity Infectious diseases Mice Molecular chains Molecular weight Polyclonal antibodies Polypeptides Positron emission Proteins S1 gene Sodium lauryl sulfate Tomography Viruses Western blotting |
| title | Cloning and Expression of Truncated Spike (Sf200) Glycoprotein of Infectious Bronchitis Virus (IBV) in Escherichia coli, and its Immunogenicity to Mice |
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