Transient expression of chicken alpha interferon gene in lettuce

Transient expression of chicken alpha interferon gene in lettuce 1 2 first-author 1 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:dgzhao@gzu.edu.cn †E-mail:dgzhao@gzu.edu.cn dgzhao@gzu.edu.cn 3 4 Guizhou Key Laboratory of Ago-Bioengineering, Guizhou University, Guiyang 550025, China Guizhou Key Laboratory of Ago-Bioengineering, Guizhou University, Guiyang 550025, China Ministry of Education Key Laboratory of Green Pesticide and Ago-Bioengineering, Guizhou University, Guiyang 550025, China Ministry of Education Key Laboratory of Green Pesticide and Ago-Bioengineering, Guizhou University, Guiyang 550025, China College of Life Sciences, Guizhou University, Guiyang 550025, China College of Life Sciences, Guizhou University, Guiyang 550025, China Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA We investigated the possibility of producing chicken alpha interferon (ChIFN-α) in transgenic plants. The cDNA encoding ChIFN-α was introduced into lettuce ( Lactuca sativa L.) plants by using an agro-infiltration transient expression system. The ChIFN-α gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus (VSV) replication on chicken embryonic fibroblast (CEF). The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry. Chicken alpha interferon (ChIFN-α) Expression Transgenic lettuce Bioactivity INTRODUCTION 1 Alpha interferon (IFN-α) possesses powerful and wide-range antiviral properties (Schultz et al., 1995a 1995a ; Ruttanapumma et al., 2005 2005 ) by regulating the pathogenesis, virulence and transmission of virus (Kochs et al., 2007 2007 ; Cauthen et al., 2007 2007 ). In response to virus, the expressions of interferon regulatory factor 3 target genes (IRF3), IFN-stimulated genes, alpha IFNs and IFN-dependent antiviral gene establish the antiviral state (Fredericksen et al., 2008 2008 ). Chicken alpha interferon (ChIFN-α) plays an important role in chicken’s defense against viral infection and the treatment of viral infection such as avian influenza virus (AIV) (Xia et al., 2004 2004 ; Wei et al., 2006 2006 ), new castle disease virus (NDV) (Marcus et al., 1999 1999 ), infectious bursal disease virus (IBDV) (Mo et al., 2001 2001 ), infectious bronchitis virus (IBV) (Pei et al., 2001 2001 ), Marek’s disease virus (MDV) (Jarosinski et al., 2001 2001 ) and Rous sarcoma virus (RSV) (Plachý et al., 1999 1999 ). Many viruses have evolved diverse mechanisms to combat the host defense mounted by IFNs (Ahmed et al., 2003 2003 ). For example, influenza virus with a full-length nonstructural protein NS1 can antagonize the induction of interferon protein and cause disease and death in chickens (Zhu et al., 2008 2008 ). Therefore, enhancing interferon-inducing capacity and increasing interferon protein levels in chicken may help to ameliorate virus pathogenicity and improve immunity of birds. It is important to produce alternative and desired broad-spectrum antiviral substance that is natural, effective, convenient and with low costs in poultry. Many viruses are threatening the health and welfare of poultry and have even struck the poultry industry and brought tremendous economic losses to the world, such as the case of the highly pathogenic avian influenza (HPAI) in recent years. At present, vaccination is the great asset for outbreak containment of avian disease (Habib et al., 2006 2006 ; Rajput et al., 2007 2007 ). However, the intensive use of vaccines is driving virus to increase its virulence such as the case of the MDV with greater virulence in vaccinated chickens (Witter, 1997 1997 ). Vaccine use is also restricted by some factors such as cryopreservation and injection technique which is difficult in immunity of migratory birds that are known to act as vectors in the dispersal of AIVs (Jourdain et al., 2007 2007 ). In addition, problems of antibiotic and drug residues in animal-derived food are a great concern of consumers. Therefore, it is wise to use naturally produced IFN in poultry industry. ChIFN-α has shown powerful antiviral activity. Nevertheless, the cost of IFN production with fermentation method or extraction from animal tissue cell is too high to be commonly used in poultry industry. Therefore, a more economic approach for IFN is needed. IFN protein from transgenic plant is obviously an attractive alternative to meet above requests. Since 1994 (Sekellick et al., 1994 1994 ), the genes encoding ChIFN-α have been successfully expressed in Escherichia coli (Schultz et al., 1995b 1995b ; Wei et al., 2006 2006 ), COS cells (Schultz et al., 1995b 1995b ) and baculovirus (Ruttanapumma et al., 2005 2005 ) but not in plants. Plants can supply a better protein secretion and low risk of contamination by animal viruses or bacterial endotoxins (Denecke et al., 1995 1995 ; Boston et al., 1996 1996 ; Ohya et al., 2001 2001 ). They are also the most economical production system of recombinant functional proteins (Khoudi et al., 1999 1999 ; Vaquero et al., 1999 1999 ). Human alpha/beta interferon ( HuIFN-α/β ) genes have been studied in cucumber, squash, lettuce and rice plants, and considerable evidence has accumulated to indicate that HuIFN-α/β can be correctly expressed in plant cells and exhibit apparent interferon activity by resistance viral infection (Zhu et al., 1994 1994 ; Arazi et al., 2001 2001 ; Sawahel, 2002 2002 ; Ohya et al., 2005 2005 ; Li et al., 2007 2007 ). Nonetheless, the feasibility of animal interferon expression has not, to our knowledge, been evaluated in plants. Poultry IFN immune system is an innovative field for antagonizing viruses and ameliorating animal health by direct oral feeding with plant feedstuff. We investigated ChIFN-α gene expression and bioactivity of recombinant protein in transgenic lettuce plants, although the expression level was low. Here we present the transformation and expression analysis of ChIFN-α by β-glucuronidase (GUS) staining, RT-PCR, ELISA and cytopathic effect (CPE) inhibition assay. MATERIALS AND METHODS 1 Transformation vector and cultivation of Agrobacterium tumefaciens 2 IFN cDNA (GenBank accession No. U07868) was amplified from plasmid pBSK-BNANSI with a ChIFN-α gene from chemical synthesis (Generay Ltd., Shanghai, China) by using the following primers: forward primer 5′-GTT CTA GAA TGG CTG TTC CAG CTT CTC-3′, and reverse primer 5′-GGG GTA CCC TAT TAC TAG GTC CTG GTG-3′. ChIFN-α gene obtained was cloned into plasmid pSH to make the plant expression vector pSFIFN-α and transfered to Agrobacterium tumefaciens strain EHA105. The resulted vector comprises cauliflower mosaic virus 35S promoter, the selectable NPT gene and report GUS gene, ChIFN-α gene ( IFNR ), Brassica napus napin signal peptide ( tp ) (Crouch et al., 1983 1983 ) and NOS terminator (Fig. 1 1 ). Recombinant Agrobacterium was maintained in a modified liquid yeast extract and beef (YEB) (Negrouk et al., 2005 2005 ) with 100 mg/L kanamycin sulfate (Amresco, USA) and 20 mg/L rifampicin (Sigma, USA). Tumefaciens was grown to log phase and condensed to an OD 600 (optical density at 600 nm) of about 2.1 for transformation of vacuum infiltration. Transformation of lettuce plants 2 Lettuce plants of the same age were harvested, rinsed with distilled water, and drained for 8 h at room temperature before vacuum infiltration. The specific transformation of lettuce was performed according to the described method by Negrouk et al.( 2005 2005 ) except that 0.065 MPa of vacuum degree was used in transformation. After infiltration, lettuce samples were cultivated for 72 h (16-h light/8-h dark photoperiod) at 22 °C (Vaquero et al., 1999 1999 ). Histochemical analysis of GUS 2 The lettuce leaves of 72 h culture were incubated with GUS assay staining solution containing the following components: 50 mmol/L NaCl, 100 mmol/L Tris-HCl (pH 7.5), 2 mmol/L potassium ferricyanide, 1 mmol/L X-gluc (Gold Biotechnology, USA) and 20% (v/v) methanol in dark at 37 °C for 3 h, and then dehydrated by 90% (v/v) ethanol. Expression detection of ChIFN-α gene by RT-PCR 2 Total RNA from the transformed lettuce leaves was used in reverse transcription and polymerase chain reaction (RT-PCR) analysis with a one-step RNA PCR kit (TaKaRa, Dalian, China) by the primers as described above. ELISA assay of ChIFN-α in lettuce leaves 2 Soluble protein was extracted for ELISA analysis by using 3 ml of extraction buffer (100 mmol/L Tris-HCl (pH 8.0), 10 mmol/L EDTA, 50 mmol/L ascorbic acid, 10.0% (v/v) glycerol, 10.0 mmol/L β-mercaptoethanol and 0.5% (w/v) SDS) per gram of leaf material. The IFN expressed was measured using a ChIFN-α ELISA kit (RapidBioLab, California, USA) following the manufacturer’s instruction. Experiments of standard curve and every sample were all repeated thrice. Chicken embryonic fibroblast IFN-α assay 2 Total soluble protein was extracted from the transgenic lettuce by using ammonium sulphate precipitation method (100% saturation) and lyophilized in a freeze dryer (Eyela, Tokyo, Japan). Biological activity of ChIFN-α expressed was measured for lethality to vesicular stomatitis virus (VSV) by using a CPE inhibition assay (Schultz et al., 1995b 1995b ). Chicken embryonic fibroblasts (CEFs) from 9-day-old chicken embryos were treated with IFN preparations from transgenic lettuce, then challenged with VSV at a 100 times 50% tissue culture infectious dose (100 TCID 50 ) after overnight culture, and continued to grow until destruction of the untreated virus-infected cells was apparent (at 48 h post-infection). The IFN titer in arbitrary units per ml (AU/ml) was expressed as a reciprocal of the dilution giving the monolayer 50% virus protection. The CPE inhibition assay was repeated thrice. RESULTS AND DISCUSSION 1 Lettuce plants of infiltration with expression vector pSFIFN-α were positively transformed in the histochemical staining reaction for GUS gene expression. ChIFN-α in the lettuce had marked mRNA expression, and there was a specific fragment near 686 bp indicating the expected gene by RT-PCR amplification (Fig. 2 2 ). The recombinant protein was found to neutralize the chook IFN-α antibody in sandwich ELISA, and the expression level was estimated to be 0.393 μg protein/kg tissue or 0.0004% of the total soluble protein according to the standard curve: Y =0.0010+0.0013 X , R 2 =0.9913, where, Y and X represent OD 450 reading and concentration of expression protein, respectively. Antiviral activity of ChIFN-α expressed by transgenic lettuce was calculated to be about 8.2×10 2 AU/mg total proteins on CEF. M: Marker; C: Plasmid; T 1 , T 2 : Different transformed lettuce plants with pSFIFN-α; M 0 : Water; M 1 : RNA of transgenic lettuce with no reverse-transcriptase; M 2 : Non-transgenic lettuce plants In this study, the antiviral ChIFN-α was first introduced into plants by a transient expression system that offers a rapid testing of the exogenous gene construction and the recombinant protein expression within a week before generating stably transformed plants (Negrouk et al., 2005 2005 ). The IFN expression was in good agreement with the experimental results of HuIFN gene in transgenic plants from others (Ohya et al., 2001 2001 ; Sawahel, 2002 2002 ; Li et al., 2007 2007 ). However, the amount of IFN expressed in this system may be not sufficient for a plant bioreactor or an oral vaccine; therefore, further studies are needed to develop a more robust system. To determine the IFN activity expressed by plants, the CEFs treated with ChIFN-α from transgenic lettuce were challenged by VSV. The results show that recombinant ChIFN-α could induce antiviral activity in CEFs, and that the maximum level of IFN activity was about 8.2×10 2 AU/mg total soluble protein, demonstrating a higher interlaboratory agreement on the expression of HuIFN-α/β gene in plant cells (Ohya et al., 2001 2001 ; Li et al., 2007 2007 ). However, the bioactivity of recombinant IFN from transgenic forage plants in animals needs to be further studied. CONCLUSION 1 In the present study, ChIFN-α was correctly transcribed and expressed in lettuce plants, and the recombinant IFN obtained was active for conferring protection against VSV infection. These findings can be valuable for prevention of infectious diseases in poultry by agricultural feed with avian cytokines. References M Ahmed , MO McKenzie , S Puckett , M Hojnacki , L Poliquin , DS Lyles . Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis . Journal of Virology , 2003 . 77 ( 8 ): 4646 - 4657 . 12663771 DOI: 10.1128/JVI.77.8.4646-4657.2003 http://doi.org/10.1128/JVI.77.8.4646-4657.2003 . Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis T Arazi , SG Slutsky , YM Shiboleth , YZ Wang , M Rubinstein , S Barak , J Yang , A Gal-On . Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits . Journal of Biotechnology , 2001 . 87 ( 1 ): 67 - 82 . 11267700 DOI: 10.1016/S0168-1656(01)00229-2 http://doi.org/10.1016/S0168-1656(01)00229-2 . Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits RS Boston , PV Viitanen , E Vierling . Molecular chaperones and protein folding in plants . Plant Molecular Biology , 1996 . 32 ( 1-2 ): 191 - 222 . 8980480 DOI: 10.1007/BF00039383 http://doi.org/10.1007/BF00039383 . Molecular chaperones and protein folding in plants AN Cauthen , DE Swayne , MJ Sekellick , PI Marcus , DL Suarez . Amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene . Journal of Virology , 2007 . 81 ( 4 ): 1838 - 1847 . 17121796 DOI: 10.1128/JVI.01667-06 http://doi.org/10.1128/JVI.01667-06 . Amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene ML Crouch , KM Tenbarge , AE Simon , R Ferl . cDNA clones for Brassica napus seed storage proteins: evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor polypeptide . Journal of Molecular and Applied Genetics , 1983 . 2 ( 3 ): 273 - 283 . 6689334 . cDNA clones for Brassica napus seed storage proteins: evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor polypeptide J Denecke , LE Carlsson , S Vidal , AS Hoglund , B Ek , MJ van Zeijl , KM Sinjorgo , ET Palva . The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo . Plant Cell , 1995 . 7 ( 4 ): 391 - 406 . 7773014 DOI: 10.1105/tpc.7.4.391 http://doi.org/10.1105/tpc.7.4.391 . The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo BL Fredericksen , BC Keller , J Fornek , MG Katze , MJr Gale . Establishment and maintenance of the innate antiviral response to west nile virus involves both RIG-I and MDA5 signaling through IPS-1 . Journal of Virology , 2008 . 82 ( 2 ): 609 - 616 . 17977974 DOI: 10.1128/JVI.01305-07 http://doi.org/10.1128/JVI.01305-07 . Establishment and maintenance of the innate antiviral response to west nile virus involves both RIG-I and MDA5 signaling through IPS-1 M Habib , I Hussain , WH Fang , ZI Rajput , ZZ Yang , H Irshad . Immunogenicity of formaldehyde and binary ethylenimine inactivated infectious bursal disease virus in broiler chicks . Journal of Zhejiang University SCIENCE B , 2006 . 7 ( 8 ): 660 - 664 . 16845721 DOI: 10.1631/jzus.2006.B0660 http://doi.org/10.1631/jzus.2006.B0660 . Immunogenicity of formaldehyde and binary ethylenimine inactivated infectious bursal disease virus in broiler chicks KW Jarosinski , W Jia , MJ Sekellick , PI Marcus , KA Schat . Cellular responses in chickens treated with IFN-alpha orally or inoculated with recombinant Marek’s disease virus expressing IFN-alpha . Journal of Interferon and Cytokine Research , 2001 . 21 ( 5 ): 287 - 296 . 11429159 DOI: 10.1089/107999001300177475 http://doi.org/10.1089/107999001300177475 . Cellular responses in chickens treated with IFN-alpha orally or inoculated with recombinant Marek’s disease virus expressing IFN-alpha E Jourdain , M Gauthier-Clerc , DJ Bicout , P Sabatier . Bird migration routes and risk for pathogen dispersion into western Mediterranean wetlands . Emerging Infectious Diseases , 2007 . 13 ( 3 ): 365 - 372 . 17552088 . Bird migration routes and risk for pathogen dispersion into western Mediterranean wetlands H Khoudi , S Laberge , JM Ferullo , R Bazin , A Darveau , Y Castonguay , G Allard , R Lemieux , LP Vézina . Production of a diagnostic monoclonal antibody in perennial alfalfa plants . Biotechnology and Bioengineering , 1999 . 64 ( 2 ): 135 - 143 . 10397849 DOI: 10.1002/(SICI)1097-0290(19990720)64:2<135::AID-BIT2>3.0.CO;2-Q http://doi.org/10.1002/(SICI)1097-0290(19990720)64:2<135::AID-BIT2>3.0.CO;2-Q . Production of a diagnostic monoclonal antibody in perennial alfalfa plants G Kochs , A García-Sastre , L Martínez-Sobrido . Multiple anti-interferon actions of the influenza A virus NS1 protein . Journal of Virology , 2007 . 81 ( 13 ): 7011 - 7021 . 17442719 DOI: 10.1128/JVI.02581-06 http://doi.org/10.1128/JVI.02581-06 . Multiple anti-interferon actions of the influenza A virus NS1 protein J Li , M Chen , XW Liu , HC Zhang , FF Shen , PW George . Transient expression of an active human interferon-beta in lettuce . Scientia Horticulturae , 2007 . 112 ( 3 ): 258 - 265 . DOI: 10.1016/j.scienta.2006.12.047 http://doi.org/10.1016/j.scienta.2006.12.047 . Transient expression of an active human interferon-beta in lettuce PI Marcus , L van der Heide , MJ Sekellick . Interferon action on avian viruses. I. Oral administration of chicken interferon-alpha ameliorates new castle disease . Journal of Interferon and Cytokine Research , 1999 . 19 ( 8 ): 881 - 885 . 10476933 DOI: 10.1089/107999099313406 http://doi.org/10.1089/107999099313406 . Interferon action on avian viruses. I. Oral administration of chicken interferon-alpha ameliorates new castle disease CW Mo , YC Cao , BL Lim . The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and new castle disease virus infection . Avian Diseases , 2001 . 45 ( 2 ): 389 - 399 . 11417818 DOI: 10.2307/1592978 http://doi.org/10.2307/1592978 . The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and new castle disease virus infection V Negrouk , G Eisner , HI Lee , K Han , D Taylor , HC Wong . Highly efficient transient expression of functional recombinant antibodies in lettuce . Plant Science , 2005 . 169 ( 2 ): 433 - 438 . DOI: 10.1016/j.plantsci.2005.03.031 http://doi.org/10.1016/j.plantsci.2005.03.031 . Highly efficient transient expression of functional recombinant antibodies in lettuce K Ohya , T Matsumura , K Ohashi , M Onuma , C Sugimoto . Expression of two subtypes of human IFN-α in transgenic potato plants . Journal of Interferon and Cytokine Research , 2001 . 21 ( 8 ): 595 - 602 . 11559437 DOI: 10.1089/10799900152547858 http://doi.org/10.1089/10799900152547858 . Expression of two subtypes of human IFN-α in transgenic potato plants K Ohya , T Matsumura , N Itchoda , K Ohashi , M Onuma , C Sugimoto . Ability of orally administered IFN-α-containing transgenic potato extracts to inhibit Listeria monocytogenes infection . Journal of Interferon and Cytokine Research , 2005 . 25 ( 8 ): 459 - 466 . 16108729 DOI: 10.1089/jir.2005.25.459 http://doi.org/10.1089/jir.2005.25.459 . Ability of orally administered IFN-α-containing transgenic potato extracts to inhibit Listeria monocytogenes infection J Pei , MJ Sekellick , PI Marcus , IS Choi , EW Collisson . Chicken interferon type I inhibits infectious bronchitis virus replication and associated respiratory illness . Journal of Interferon and Cytokine Research , 2001 . 21 ( 12 ): 1071 - 1077 . 11798465 DOI: 10.1089/107999001317205204 http://doi.org/10.1089/107999001317205204 . Chicken interferon type I inhibits infectious bronchitis virus replication and associated respiratory illness J Plachý , KC Weining , E Kremmer , F Puehler , K Hala , B Kaspers , P Staeheli . Proctive effects of type I and type II interferons toward Rous sarcoma virus-induced tumors in chickens . Virology , 1999 . 256 ( 1 ): 85 - 91 . 10087229 DOI: 10.1006/viro.1999.9602 http://doi.org/10.1006/viro.1999.9602 . Proctive effects of type I and type II interferons toward Rous sarcoma virus-induced tumors in chickens ZI Rajput , CW Xiao , SH Hu , AG Arijo , NM Soomro . Improvement of the efficacy of influenza vaccination (H5N1) in chicken by using extract of Cochinchina momordica seed (ECMS) . Journal of Zhejiang University SCIENCE B , 2007 . 8 ( 5 ): 331 - 337 . 17542061 DOI: 10.1631/jzus.2007.B0331 http://doi.org/10.1631/jzus.2007.B0331 . Improvement of the efficacy of influenza vaccination (H5N1) in chicken by using extract of Cochinchina momordica seed (ECMS) R Ruttanapumma , M Nakamura , K Takehara . High level expression of recombinant chicken interferon-alpha using baculovirus . The Journal of Veterinary Medical Science , 2005 . 67 ( 1 ): 25 - 28 . 15699590 DOI: 10.1292/jvms.67.25 http://doi.org/10.1292/jvms.67.25 . High level expression of recombinant chicken interferon-alpha using baculovirus WA Sawahel . The production of transgenic potato plants expressing human alpha-interferon using lipofectin-mediated transformation . Cellular and Molecular Biology Letters , 2002 . 7 ( 1 ): 19 - 29 . 11944047 . The production of transgenic potato plants expressing human alpha-interferon using lipofectin-mediated transformation U Schultz , B Kaspers , C Rinderle , MJ Sekellick , PI Marcus , P Staeheli . Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity . European Journal of Immunology , 1995 . 25 ( 3 ): 847 - 851 . 7535703 DOI: 10.1002/eji.1830250332 http://doi.org/10.1002/eji.1830250332 . Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity U Schultz , C Rinderle , MJ Sekellick , PI Marcus , P Staeheli . Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active . European Journal of Biochemistry , 1995 . 229 ( 1 ): 73 - 76 . 7538075 DOI: 10.1111/j.1432-1033.1995.0073l.x http://doi.org/10.1111/j.1432-1033.1995.0073l.x . Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active MJ Sekellick , AF Ferrandino , DA Hopkins , PI Marcus . Chicken interferon gene: cloning, expression, and analysis . Journal of Interferon Research , 1994 . 14 ( 2 ): 71 - 79 . 7521382 . Chicken interferon gene: cloning, expression, and analysis C Vaquero , M Sack , J Chandler , J Drossard , F Schuster , M Monecke , S Schillberg , R Fischer . Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves . Proceedings of the National Academy of Sciences , 1999 . 96 ( 20 ): 11128 - 11133 . DOI: 10.1073/pnas.96.20.11128 http://doi.org/10.1073/pnas.96.20.11128 . Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves Q Wei , GQ Peng , ML Jin , YD Zhu , HB Zhou , HY Guo , HC Chen . Cloning, prokaryotic expression of chicken interferon-alpha gene and study on antiviral effect of recombinant chicken interferon-alpha . Sheng Wu Gong Cheng Xue Bao , 2006 . 22 ( 5 ): 737 - 743 . (in Chinese) 17037195 . Cloning, prokaryotic expression of chicken interferon-alpha gene and study on antiviral effect of recombinant chicken interferon-alpha RL Witter . Increased virulence of Marek’s disease virus field isolates . Avian Diseases , 1997 . 41 ( 1 ): 149 - 163 . 9087332 DOI: 10.2307/1592455 http://doi.org/10.2307/1592455 . Increased virulence of Marek’s disease virus field isolates C Xia , J Liu , ZG Wu , CY Lin , M Wang . The interferon-α genes from three chicken lines and its effects on H9N2 influenza viruses . Animal Biotechnology , 2004 . 15 ( 1 ): 77 - 88 . 15248602 DOI: 10.1081/ABIO-120037900 http://doi.org/10.1081/ABIO-120037900 . The interferon-α genes from three chicken lines and its effects on H9N2 influenza viruses Q Zhu , H Yang , W Chen , W Cao , G Zhong , P Jiao , G Deng , K Yu , C Yang , Z Bu , 等 . A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens . Journal of Virology , 2008 . 82 ( 1 ): 220 - 228 . 17942562 DOI: 10.1128/JVI.00978-07 http://doi.org/10.1128/JVI.00978-07 . A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens Z Zhu , KW Hughes , L Huang , BL Sun , CM Liu , Y Li . Expression of human α-interferon cDNA in transgenic rice plants . Plant Cell Tissue and Organ Culture , 1994 . 36 ( 2 ): 197 - 204 . DOI: 10.1007/BF00037720 http://doi.org/10.1007/BF00037720 . Expression of human α-interferon cDNA in transgenic rice plants 10.1631/jzus.B0710596 Project supported by the National Key Technology Research and Development Program of China (No. 2007BAD59B06), the International Science and Technology Cooperation Program of China (No. 2007DFA31260), and the Science Foundation for the Excellent Youth Scholars of Guizhou Province, China (No. 20030312) 2007-11-20 2008-03-18 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 09