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Construction of Three Nucleic Acid Vaccines of Swine Hepatitis E Virus ORF2 Ger\e Continuous Fragment

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Animal Husbandry and Feed Science 2015年第5期7卷 308-313页

作者： Li Bin su qianlian Zhao Wu Qin Yibin Liang Jiaxing Xiao Aihuan Lu Bingxia Li Yingying He Ying Duan Qunpeng Jiang Jiajia Liang Baozhong Guangxi Veterinary Research Institute Guangxi Key Laboratory of Animal Vaccines and New Technology College of Animal Science and Technology Guangxi University

In order to develop swine hepatitis E （HE） genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.

关键词： Swine Hepatitis E virus ORF2 Eukaryotic expression plasmid Nucleic acid vaccine

Detection and Analysis of Routine Blood Indexes in Guike Pig

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Agricultural Biotechnology 2018年第3期7卷 95-98,103页

作者： qianlian su Bin LI Jiaxing LIANG Yingning ZHOU Zhongwei CHEN Yibin QIN Bingxia LU Ying HE Qunpeng DUAN Dongfu JIANG Jingzhuan LU Wu ZHAO Guangxi Veterinary Research Institute/Guangxi Key kahoratory of Veterinary Biotechnology Nanning 530001 China

[Objectives] In this study,routine blood indexes of Guike pig were detected to clarify quantity variation and morphology distribution of different functional cells（ red blood cells,white blood cells and platelets）,thus providing reference data for immunological evaluation and disease resistance research of Guike pig. [Methods] Blood samples were collected from forty-four 49-day-old healthy Guike pigs（ 22 males and 22 females） once a week for seven consecutive weeks to detect routine blood indexes,and the results were compared with routine blood indexes of Landrace pig and Enshi black pig. [Results]Among red blood cell indexes of Guike pig at different day ages,red blood cell count（ RBC） and hemoglobin count（ HGB） both reached the lowest levels（ 6. 85 × 10^12/L and 97. 1 g/L）at 70 days of age,which were significantly lower than the corresponding indexes at 49,56,63,84 and 91 days of age（ P 〈 0. 05,similarly hereinafter）; mean corpuscular hemoglobin（ MCH） and mean corpuscular hemoglobin concentration（ MCHC） demonstrated basically the same variation pattern,which both reached the lowest levels（ 15. 11 pg and 275. 70 g/L） at 70 days of age and reached the highest levels（ 16. 40 pg and 314. 30 g/L） at 91 days of age; hematocrit（ HCT）,mean corpuscular volume（ MCV） and red blood cell distribution width（ RDW） exhibited relatively stable changes with no significant differences among different day ages（ P 〉 0. 05,similarly hereinafter）. Among white blood cell indexes of Guike pig at different day ages,white blood cell count（ WBC） reached the lowest level（ 13. 71 × 10^9/L） at 63 days of age and reached the highest level（ 17. 52 × 10^9/L） at 84 days of age,with significant differences; neutrophil count（ NEUT）reached the lowest level（ 3. 61 × 10^9/L） at 70 days of age,which was significantly lower than that at 49,56 and 77 days of age; lymphocyte count（ LYMPH） exhibited relatively stable changes and was not significantly affected by the increase in day age. Among platelet indexe

关键词： Guike pig Landrace pig Enshi black pig Routine blood indexes Detection and analysis

Phylogenetic Analysis of HN Gene of Eight Pigeon NDV Isolates in Guangxi

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Animal Husbandry and Feed Science 2018年第2期10卷 136-138页

作者： Lu Bingxia Liang Jiaxing Duan Qunpeng Chen Zhongwei Jiang Dongfu Lu Jingzhuan Zhou Yingning Bi Bingfen He Ying Qin Yibin Li Bin su qianlian Zhao Wu Guangxi Key Laboratory of Veterinary Biotechnology Guangxi Veterinary Research Institute

[Objective] The paper was to provide a basis for scientific prevention and control of pigeon Newcastle disease（ND）.[Method] The HN gene of eight pigeon NDV strains isolated from different pigeon farms in Guangxi were amplified by RT-PCR,sequenced and *** molecular evolution characteristics of HN gene of pigeon NDV isolates in Guangxi was discussed.[Result] The nucleotide sequence length of HN gene of the eight NDV isolates was 1 716 bp,encoding 571 amino *** belonged to virulent group C,and the gene length characteristic of HN gene accorded with virulent *** of nucleotide homologies indicated that the eight NDV isolates shared higher homology with genotype VIb,ranging from 90.4% to 99.5%.Phylogenetic tree analysis demonstrated that the genetic relationship between the eight NDV strains in Gangxi and the NDV isolates from Guangxi,Guangdong,Jilin,Liaoning,Yunnan and Heilongjiang during 2011 and 2013 was *** were located in the same cladogram branch.[Conclusion] We assume that the eight pigeon NDV isolates in Guangxi all belong to the gene class II genotype VI b NDV.

关键词： Pigeon Newcastle disease virus (NDV) HN gene Phylogenetic analysis

Construction and Preliminary Application of RT-LAMP Detection Method for Swine Japanese B Encephalitis Virus

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Agricultural Biotechnology 2013年第4期2卷 38-41,46页

作者： Bingxia LU Lei LIU Yibin QIN Wu ZHAO Jiaxing LIANG Xuan GUO Mulan LI Bin LI Ying HE qianlian su Baozhong LIANG Bingfen BI Weijian HUANG Guangxi Veterinary Research Institute College of Animal Science and Technology Guangxi University

[ Objective] This study aimed to establish a rapid, sensitive and specific method using reverse transcription loop-mediated isothermal amplification （RT-LAMP） technology to detect swine Japanese B encephalitis virus （JEV）. [ Method ] Four specific LAMP primers were designed according to six loci the conservative region of JEV E gene sequence. Positive JEV RNA sample was used as a template for one-step amplification, and the reaction conditions and reaction system were optimized. [ Result] Experimental results showed that the established method had high sensitivity, with the detection limit of 0.5pg; specificity experi- ments indicated that the method had high specificity and there was no amplification reaction for other viral pathogens. The coincidence rate between detection results of RT-LAMP and RT-Nested-PCR was 90.9%. After RT-LAMP reaction, a chemiluminescent agent was added for visual observation, which greatly reduced the detection time. This method required no special equipment but only a water bath, which was a simple, sensitive and rapid detection method for swine Japanese B encephalitis virus and could be applied in primary laboratories. [ Conclusion] An RT-LAMP detection method for swine Japanese B encephalitis virus was successfully established and preliminarily applied in clinical practice.

关键词： Swine Japanese B encephalitis virus RT-LAMP Sensitivity Specificity

Detection of Porcine Epidemic Diarrhea Virus in Guangxi Province from 2011 to 2014 and Sequence Analysis of Its M Gene

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Animal Husbandry and Feed Science 2016年第1期8卷 12-17,38页

作者： Lu Bingxia Qin Yibin He Ying Li Yingying Liang Jiaxing Li Keyu Li Bin su qianlian Zhou Yingning Jiang Dongfu Lu Jingzhuan Bi Bingfen Liang Baozhong Duan Qunpeng Zhao Wu Guangxi Key Laboratory of Animal Vaccines and New Technology Guangxi Veterinary Research Institute College of Animal Science and Technology Guangxi University

Detection of pigs epidemic diarrhea virus （PEDV） was conducted on 331 piglets diarrhea fecal samples collected in Nanning, Yulin and other 12 areas of Guangxi Province from January of 2011 to April of 2014 by the method of reverse transcription-polymerase chain reaction （RT-PCR）. The results showed that the positive samples of PEDV were 210 and the positive rate was 63.44%. The clone and sequencing of M gene was carried out on 25 positive samples. PEDV reference strains were selected from GeneBank to conduct the sequence homology alignment analysis and the phylogenetic tree of M gene. The M gene homology and amino acid sequence identity between 25 isolated strains and 51 reference strains were 96.0% - 99.6% and 94.3% - 99.6%, respectively. The genetic variation anal- ysis of M gene showed that the genetic relationship of PEDV prevalent strains in Guangxi Province from 2013 to 2014 was close to that of the prevalent strains in Bei- jing, Anhui, Wuhan, Hebei and Guangdong from 2010 to 2013, and which were far from that of the Chinese early isolates CH/S （GenBank number： JN547228 ）, vaccine strain CV777 （GenBank number： AF353511 ） and Attenuated DR13 （GenBank number： JQ023162）. Indicating that the PEDV strains prevalent in Guan- gxi in recent years showed significant variation with the early isolates.

关键词： Porcine epidemic diarrhea virus (PEDV) M gene Genetic variation

Isolation of Swine Corynebacteria and Analysis of 16S rDNA Gene

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Animal Husbandry and Feed Science 2016年第6期8卷 339-343页

作者： Qin Yibin He Pingping Lu Bingxia He Ying Li Bin Liang Jiaxing Duan Qunpeng Chen Zhongwei Bi Bingfen Zhou Yingning su qianlian Jiang Dongfu Li Keyu Lu Jingzhuan Zhao Wu Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Animal Vaccines and New Technology Nanning 530001 China Guangxi Institute of Fishery Sciences Nanning 530021 China College of Animal Science and Technology Guangxi University Nanning 530005 China

A strain of gram-positive bacillus was isolated from suppurative lung organs of nursery pigs in a pig farm, which was further characterized by morphological observation, cultivation test, biochemical test, drug sensitivity test, pathogenicity test and 16S rDNA gene cloning and sequence analysis. The results showed that the isolate grew well in rabbit blood agar plate and horse serum tryptone soybean agar （TSA） plate under aerobic condition, which could lead to mortality of mice and were susceptible to cephalosporin antibiotics and fluoroquinolone antibiotics. Phylogenetic analysis showed that the isolate had close genetic evolutionary relationships with Corynebacterium bacteria, and the sequence of 16S rRNA gene shared the homology of 91.7% -98.3% with the representative strain of coryne- bacteria, indicating the isolated strain was eorynebactefium.

关键词： Coryrtebacterium Isolation and identification 16S rRNA Gene analysis

猪细小病毒自然弱毒N株VP2基因的克隆、测序及生物信息学分析

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基因组学与应用生物学 2010年第5期29卷 849-856页

作者：李斌梁家幸赵武苏乾莲何颖梁保忠秦毅斌何苹萍广西兽医研究所南宁530001

本研究对猪细小病毒(porcine parvovirus,PPV)自然弱毒N株(PPV-N)VP2基因进行克隆、测序并利用生物信息学技术分析PPV-NVP2蛋白基因的同源性、遗传进化、密码子偏爱性、糖基化位点、磷酸化位点、B细胞抗原表位及其二、三级结构。结果表... 详细信息

本研究对猪细小病毒(porcine parvovirus,PPV)自然弱毒N株(PPV-N)VP2基因进行克隆、测序并利用生物信息学技术分析PPV-NVP2蛋白基因的同源性、遗传进化、密码子偏爱性、糖基化位点、磷酸化位点、B细胞抗原表位及其二、三级结构。结果表明:成功扩增出包含VP2基因完整目的片段(1901bp),构建了VP2基因的克隆重组质粒pMD18-T-VP2,测序获取VP2基因序列(1740bp)并将该序列登录到GenBank(HM355807)。PPVVP2基因属高度保守的基因;PPV-N株与PPV弱毒代表毒株NADL-2株亲缘性近,推测PPV-N株属于弱毒株;PPV-N株VP2基因氨基酸密码子偏爱以A结尾的密码子;PPV-N株VP2蛋白可能存在7个糖基化位点,其丝氨酸、苏氨酸和酪氨酸可能分别有9、7、8个磷酸化位点,可能存在24个B细胞抗原表位;PPV-N株VP2蛋白二级结构预测,α-螺旋占11.74%,β-折叠占22.97%,无规则卷曲占65.28%,而三维结构预测VP2蛋白主要以无规则卷曲为主,存在多个螺旋和折叠区域。本研究结果为进一步阐释PPV-N株自然弱毒的分子机理提供依据,并为PPV分子诊断试剂及基因工程疫苗研究等提供有益借鉴。

关键词： PPV-N株 VP2基因克隆测序生物信息学分析

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广西14市猪戊型肝炎的分子流行病学分析

基因组学与应用生物学 2010年第3期29卷 464-470页

作者：赵武秦毅斌肖爱欢梁家幸何颖李斌刘芳苏乾莲梁保忠黄伟坚广西兽医研究所南宁530001 广西大学动物科学技术学院南宁530005

为调查广西猪戊型肝炎(HE)的感染情况,本研究应用套式RT-PCR方法对采自广西14市43个猪场的508份3月龄左右猪只粪样及市售182份猪肝扩增猪戊型肝炎病毒(HEV)ORF2基因特异序列,并进行核苷酸序列同源性分析及遗传进化分析。结果表明,在508... 详细信息

为调查广西猪戊型肝炎(HE)的感染情况,本研究应用套式RT-PCR方法对采自广西14市43个猪场的508份3月龄左右猪只粪样及市售182份猪肝扩增猪戊型肝炎病毒(HEV)ORF2基因特异序列,并进行核苷酸序列同源性分析及遗传进化分析。结果表明,在508份猪粪样中194份可以检测到HEV RNA,阳性率为38.2%;182份猪肝样本中,33份可检测到HEV RNA,阳性率为18.1%;43个猪场中检出HEV RNA的有35个,猪场阳性率为81.4%。所获取的39株广西猪HEV ORF2基因特异序列同源性为89.9%～100%,提示在所调查的广西猪场中已较普遍存在HEV感染,所获取的39株广西HEV阳性样品均属于基因Ⅳ型。本研究结果佐证了感染猪及市售带毒猪肝脏是HEV重要的储存库和传染源,应该重视其公共卫生学意义,预防粪-口途径或者食源性HEV感染。

关键词：猪戊型肝炎戊型肝炎病毒分子流行病学基因型

猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析

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中国畜牧兽医 2018年第1期45卷 178-188页

作者：秦毅斌卢冰霞何颖段群棚李斌陈忠伟梁家幸周英宁苏乾莲蒋冬福赵武广西兽医研究所广西兽医生物技术重点实验室南宁530001

本研究旨在获得可在细胞培养中稳定、有效生长增殖的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)分离毒株,并对其全基因组序列进行测定分析。应用Vero细胞从广西腹泻仔猪肠道内容物中进行病毒分离,通过细胞病变和RT-PCR对... 详细信息

本研究旨在获得可在细胞培养中稳定、有效生长增殖的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)分离毒株,并对其全基因组序列进行测定分析。应用Vero细胞从广西腹泻仔猪肠道内容物中进行病毒分离,通过细胞病变和RT-PCR对细胞培养物进行鉴定,应用下一代测序技术对分离毒株全基因组序列进行测定。结果显示,成功分离到1株PEDV,命名为CH/GX/2015/750A。该毒株可稳定有效地在Vero细胞生长增殖,并引起典型的细胞病变;已在Vero细胞连续传代25代,病毒滴度随着传代次数的增加逐渐提高并稳定在107.50TCID50/mL。该毒株全基因组序列长28 038bp;与22个参考毒株的全基因序列比对显示,核苷酸同源性为96.8%~99.8%,其中与YC2014株同源性最高,为99.8%。全基因组和S基因系统进化分析显示,PEDV CH/GX/2015/750A分离毒株属于Ⅱa亚群,与YC2014、PEDV-WS等变异毒株亲缘关系密切。结果表明,本研究分离获得的CH/GX/2015/750A毒株是PEDV地方流行变异毒株。

关键词：猪流行性腹泻病毒(PEDV) 分离鉴定全基因序列分析