Cui Bao-an

Henan Agricultural University

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Hong‐Ying Chen

Henan Agricultural University

Zhanyong Wei

Henan Agricultural University

Mingfan Yang

Henan Agricultural University

Hongying Zhang

Nanchang University

Xinsheng Li

Zhejiang Normal University

Pingan Xia

Henan Agricultural University

Yabin Wang

Beijing Institute of Technology

Zheng LanLan

Henan Agricultural University

Hongying Chen

Sichuan University

Xuebing Wang

Hokkaido University

Cooperative Institutions

Henan Agricultural University

Chinese Academy of Sciences

Chinese Academy of Medical Sciences & Peking Union Medical College

Nanjing Agricultural University

Sun Yat-sen University

Central South University

Fudan University

Huazhong University of Science and Technology

Shanghai Jiao Tong University

Sichuan University

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Effects of isatis root polysaccharide on proliferation and excretion of cytokines and NO of swine splenic lymphocytes in vitro

He'nan nongye daxue xuebao (2009)

Cui Bao-an

The study was conducted to investigate the mechanism of immuno-regulation induced by isatis root polysaccharide(IRPS).The effects of the IRPS on proliferation of swine splenic lymphocytes in vitro was tested by the method of MTS colorimetric assay.NO was detected by reduction of nitrate method and IL-2,IL-4,IFN-γ were detected by ELISA.The results showed that IRPS could stimulate proliferation of swine splenic lymphocytes induced by ConA or LPS significantly(P0.05)in vitro.Upregulation of IFN-γ co-induced by ConA and IRPS in all three dose groups were observed.In contrast,IRPS appeared to inhibit the secretion of IL-2 induced by ConA and had no influence on IL-4 induced by ConA.The level of NO secreted by swine splenic lymphocytes was enhanced significantly(P0.05)by IRPS in the low dose group.

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Identification and determination of virulence gene for different isolates of E. faecalis originated from infective piglet

Zhongguo shouyi xuebao (2010)

Yabin Wang Liying Chen Hongying Zhang Lei Liu Cheng JinPing

Among the recently isolated Enterococcal strains from some infected swine flocks in Henan province,five representatives of different origins were selected for systemic identification including pathogenicity assessment and virulent gene detection.The results indicated that the pathogen that caused swine infection was Enterococcus faecalis.The isolates showed similar morphological and cultivating characteristics and tolerance of extreme environments,whilst performed differently in carbohydrate fermentation;sensitive to vancomycin,teicoplanin,rifampicin and ampicillin,whereas totally tolerant to common clinical drugs such as erythromycin,kanamycin and tetracycline.The 16S rRNA analysis indicated that these isolates possess high homology to E.faecalis ATCC29212 (99.6%-99.8%),and the nucleotide homology to E.faecalis NC_004668 and AJ301831 strains that are published in GenBank are 99.7%-99.9% and 99.5%-99.7% respectively.Through analysis of virulence phenotype and six main virulent genes and determination of LD50 to mice,we discovered that the five isolates carried different virulent genes,and the HE1 and HE2 strains which carried all the tested genes were the most pathogenically potential,yet the 4-gene carrying HE41 was least capable.The challenge tests were respectively performed with piglets aged 20d with isolates of HE1 and HE5 and all piglets tested were infected and showed clinical symptoms.

Teicoplanin

Enterococcus faecalis

Kanamycin

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Cloning and expression of NP gene of H9N2 subtype avian influenza virus henan isolate in Escherichia coli

Journal of Northwest A&F University (2009)

Hongying Chen Fang ZhongYi Cui Bao-an Xinsheng Li Pingan Xia

【Objective】 The research was to study cloning and expression of nucleoprotein(NP) gene of H9N2 subtype avian influenza virus(AIV) henan isolate in Escherichia coli.【Method】 A pair of specific primers were designed and synthesized according to NP gene sequences published in GenBank.NP cDNA was amplified by reverse transcription-polymerase chain reaction(RT-PCR) from the virus RNA directly extracted from chicken embryo allantoic fluid infected by H9N2 subtype AIV henan isolate,and cloned into pGEM-T easy vector and sequenced.The NP gene segment which encoded the major epitopes of nucleoprotein was subcloned into prokaryotic expression vector pET-28a(+) using PCR and enzyme digestion.The resulting plasmid,pET-28 NP,was used to transform into E.coli BL21(DE3),which can highly express recombinant NP protein by using IPTG induction.【Result】 The results showed that the nucleotide sequence of NP gene was 1 497 bp in length,which included one open-reading frame,encoding 498 amino acid residues.When the nucleotide sequence of NP gene of henna isolate was compared with that of 7 strains of AIV in GenBank,homology was higher than 95% at nucleotide level,and the deduced amino acid homology was higher than 98%.The expressed product was identified by SDS-PAGE and Western blot.The results showed that recombinant protein was about 45 ku,and can highly be expressed in E.coli BL21(DE3) by using 1.0 mmol/L IPTG induction for 5 h.The recombinant protein can react with sera against H9 subtype AIV.【Conclusion】 NP gene for H9N2 AIV was highly reserved.Recombinant NP protein for H9N2 AIV can be expressed in expression system pET-28NP and had good immunogenicity.

Nucleoprotein

Homology

Cloning (programming)

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Expression of gD gene of infectious laryngotracheitis virus and primary development of indirect ELISA assy based on the expressed protein.

Zhongguo shouyi xuebao (2009)

Mingfan Yang Cui Bao-an Sumei Zhang Hongying Chen LI Xiang-rui

The gD gene of infectious laryngotracheitis virus was obtained by digesting recombinant plasmid pGEM-T-gD with EcoR Ⅰ and Hind Ⅲ and then cloned into pET-28a(+)to obtain the expression plasmid designated as pET-gD.The pET-gD was further transformed into E.coli BL21 cells and a 55 000 protein was high expressed after induced with IPTG.Western-blotting analysis proved the recombinant protein has a good reactive ability against ILTV positive serum.The indirect ELISA for detecting ILTV antibodies was established by using purified recombinant gD protein.The optional working circumstances for the ELISA(antigen concentration of 24.5 mg/L,serum dilution of 1∶80) were tried out with chess titration.The tested serum of positive criterion of this ELISA is D450≥0.25,the negative serum D4500.25.

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Prokaryotic Expression of GP2 Protein of Porcine Reproductive and Respiratory Syndrome Virus Hn-1/06 Strain

Guangdong nongye kexue (2009)

Cui Bao-an

A pair of specific primers of porcine reproductive and respiratory syndrome virus(PRRSV) ORF2 were designed and synthesized according to the published gene sequence of PRRSV from GenBank(Accession number: PRU87392),and ORF2 gene of PRRSV Hn-1/06 strain was obtained by RT-PCR method.The RT-PCR product was directly cloned into pTG19-T vector and this recombinant plasmid was identified by enzyme digestion and DNA sequencing.The positive clone was designated pTG19-T-GP2.After being cloned into the prokaryotic expression vector pGEX-6p-1,the ORF2 gene was successfully obtained with the induction of IPTG.Western-blot test result confirmed that the expressed fusion protein could specifically react with antiserum against PRRSV.

Strain (injury)

clone (Java method)

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Preparation of Oleum curcumae aromaticae oil injection and its in vitro effects on anti-PRRSV.

He'nan nongye kexue (2010)

XU Duan-hong Cui Bao-an Xuebing Wang Hongying Zhang Zhanyong Wei

For the screening of medicines anti-porcine reproductive and spiratory syndrome virus(PRRSV),Oleum curcumae aromaticae oil injection was prepared as the investigational drug to observe its in vitro effect on PRRSV.For a negative control,ribavirin injection was used.The results showed that,since the maximum safe concentration to do multiple proportion dilute,they had a certain degree of inhibition,killing,and blocking effects on PRRSV.Among them,the killing effect of oleum curcumae aromaticae oil injection was more prominent and ribavirin injection had a significant effect on inhibitory action and killing.

Oleum

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Gene cloning, sequence analysis and prokaryotic expression vector construction of duck MHC-I α chain mature peptide of the extracellular domain.

Journal of Northwest A&F University (2010)

Kai Sun Xinsheng Li Cui Bao-an Hong‐Ying Chen Zhanyong Wei

【Objective】The study was done to clone duck MHC-Ⅰα chain mature peptide gene cDNA sequence of the extracellular domain and construct a prokaryotic expression plasmid.【Method】The cDNA of duck MHC-Ⅰα chain extracellular domain of mature peptide gene amplified with RT-PCR from healthy duck spleen tissue was cloned into pGEM-T Easy vector by T/A Ligation.After double digestion,MHC-Ⅰα chain extracellular domain of mature peptide cDNA fragment was subcloned into pET-28a(+) vector to construct a prokaryotic expression plasmid.Restriction endonucleases analysis and sequencing were used to conform the recombinant plasmid.【Result】The duck MHC-Ⅰα chainmature peptide gene of the extracellular domain is 813 bp in length encoding 200 amino acids.The gene shared 89.4%-91.7% nucleotide homology and 78.2%-83.7% amino acid homology with other duck MHC-Ⅰα chain mature peptide gene of the extracellular domain.Phylogenetic tree analysis indicated that the duck MHC-Ⅰα chain mature peptide gene sequence variation was related to genus,the nearer relationship,the higher homology.The duck MHC-Ⅰα chain mature peptide gene of the extracellular domain was correctly inserted into prokaryotic expression vector.【Conclusion】The result showed that the recombinant plasmid pET/Du MHC-Ⅰ was constructed correctly,which paved the way for further studies on the MHC-Ⅰ protein expression and its biological activities.

Homology

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Prokaryotic expression and the recombinant protein renaturation of the M gene of porcine reproductive and respiratory syndrome virus Hn-1/06 strain

He'nan nongye daxue xuebao (2009)

Cui Bao-an

The gene segments tM deleting hydrophobic region sequence were successfully amplified from recombinant plasmids pTG19-T-M by PCR.The fragment was inserted into the prokaryotic expression vector pET-32a to construct the recombinant plasmid pET-tM,and expressed in E.Coli BL21.The result indicated that the fusion protein pET-tM was successfully expressed.SDS-PAGE analysis demonstrated the molecular weight of reorganization protein existing in cytorrhyctes type was 29 kD and amounted to 31% of the mass of bacterial proteins in cvtorrhvctes.The renaturation of reorganization protein was carried on by dilution and the dialysis after treatment with the 8 mol·L-1 urea.The renaturation reorganization protein was analyzed by Western-blot,the result demonstrated that the renaturation reorganization protein was recognized by the PRRSV masculine blood serum.

Molecular mass

Strain (injury)

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The Analysis of Antiviral Cytokines Transcriptional Profiles of PK-15 Cell Cultures Following Infection with Porcine Parvovirus

Xumu shouyi xuebao (2011)

Cui Bao-an

In order to survey the host inflammatory responses,particularly the antiviral cytokines responses of Porcine Parvovirus(PPV) infection,and to investigate the host-PPV interaction,Porcine Kidney-15 cells(PK-15) were infected by PPV.Then the viral DNA was measured and analyzed using real-time PCR.The transcript level of cytokines(IFN-β,IFN-γ,IFNAR-1,IFNAR-2,MHC-Ⅰ,MHC-Ⅱ,MX1,NOS,2-5AS,RNase L and IRF-3) were detected by real-time PCR too.The results showed that PPV proliferated rapidly in PK-15 cell 12 hours after infection,and reached peak 48 hours after infection;the transcription levels of IFN-β,IFN-γ,IFNAR-1,IFNAR-2,MHC-Ⅰ,MHC-Ⅱ,MX1,iNOS,2-5AS,RNase L and IRF-3 were increased obviously,and the transcription level of Mx1 gene was increased 8 423 times at 24 hours after infection.The results revealed that the level of antiviral cytokines increased in PPV infected PK-15 cell.

Porcine parvovirus

Transcription

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Amplification,Cloning and Sequencing of Interferon-α Gene from Poodle

Jiangsu nongye xuebao (2010)

Song YaPeng Hong‐Ying Chen Cui Bao-an Yanyan Jia Guo XianPo

One pair of primers was designed and synthesized according to the canine interferon-α(IFN-α) gene nucleic acid sequence published in the GenBank.The IFN-α gene was amplified from genome DNA of poodle liver by PCR.The purified PCR products were linked to pGEM-T Easy vector and then transformed into competent cells of DH5α.By identification of blue-white spot screening,plasmid PCR and enzyme digestion,positive strain was sent to TaKaRa for sequencing.The sequencing results showed that poodle IFN-α gene was obtained.The cloned poodle IFN-α gene span is 564 bp,which includes one open-reading frame encoding 187 amino acid residues.And there are three potential N-glycosalation sites and ten cysteines in the deduced amino acid sequence.Homology of nucleotide and amino aids of IFN-α gene between poodle and humans and other animals and the phylogenetic tree indicated that IFN-α gene varied in species,and the closer the relationship,the higher the homology.

Homology

Cloning (programming)

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