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Proviral Genomic Variation of EIAV Envelop gp 90 Gene Observed in Experimentally Infected Horse

Quankai WANG ; Hai PANG ; Xiangang KONG ; Hua XUAN ; Jingang ZHANG ; Jingliang LU ; Enge FEI

Chinese Journal of Veterinary Science.1999;19(3):207-211.

The present experiment was designed to observe the genetic variation of equine infectious anemia virus (EIAV) envelop gp 90 gene in infected horse. One horse was infected experimentally with P337-V70 strain and showed no clinical signs after being infected at twice with the same virus strain. Seventeen proviral sequences covering principal neutralizing domain (PND) of EIAV gp 90 gene were obtained from the buffy coat and liver of the horse through PCR amplification and cloning. Comparative analysis of the sequences revealed that some sequences contained the nucleotide insertions in the PND region. The insertions might be generated by direct repeat and strand displacement of sequence segment in their PND gene, showing different lenghts.

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Antibiotic Sensitivity Detection and Analysis of Escherichia coli Isolated Clinically in China

Liancheng LEI ; Dan ZHENG ; Wenyu HAN ; Fengtao JI ; Weidong XU

Chinese Journal of Veterinary Science.2005;25(5):470-473.

The susceptibility to 9 kinds of antibiotics of 393 animal pathogenic E. coli isolated from clinical samples was determined from 2000 to 2003. The resistance to TC, GM, CMP, AMP, RA, KM, FT, SM and CIP were 93.89%, 57.76%,78.63%, 77.86%, 92.11% ,47.33%, 46.82%, 76.84% and 74.81%, respectively. The isolates could be classified into eight classes according to the number of drugs to which srains were resistant. The resistance spectrum of the isolates varied from 2to 9 kinds of the above drugs. The strains that were resistant to seven kinds of drugs were more than 80 percent. The resistance rates of swine origin strains to GM,AMP and KM were higher than those of poultry origin strains, while the resistance rates of poultry origin strains to TC, CMP, SM and CIP were higher remarkably than those of swine origins. The frequency of resistance increased from 2000 to 2003,which was from 67.33% to 90.58% for CMP, 71.29% to 84.81% for AMP, 73.76%to 80.10% for SM and 61.88% to 88.48% for CIP. At the same time, the resistance rate of GM decreased from 61.39% to 53.92%. Thirty-seven strains (95 %) could make all the Kunming mice die within 72 h injected intraperitoneally with the culmice three times in vivo. The pathogenicity of wild strains with drug resistance acquired naturaly to mice did not decline.

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Expression of Bovine Interleukin-2 Gene in Pichia pastoris

Fang CHEN ; Hongli SUN ; Xiangrong CAO ; Zhen LI ; Ruisong YU

Chinese Journal of Veterinary Science.2005;25(2):178-182.

The Interleukin-2 gene cDNA was cloned into the Pichia pastoris expression vector pPICZB,which is under the control of the alcohol oxidase promoter AOX1. The linearized recombinant plasmid of BoIL2-pPICZB,digested by Sac I ,was transformed into X-33 strains by electroporation. The multi-copy insert transformants were screened by Zeocin-resistance and induced by 1% methanol. The intracellular expression products were tested by SDS-PAGE analysis and Western blotting. Purified recombinant BoIL2 was gained by metal-chelating affinity chromatographic (MCAC). Assay with murine CTLL-2 cells showed that the recombinant BoIL2 exhibited the biological activity.

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The Influence of Sera,Gonadotropins,EGF and Water on In vitro Maturation of Goat Oocytes

Chao WANG ; Zhiqing WEI ; Xiangchen LI ; Zhixin AN ; Zhiping ZHANG ; Jianhong SHU ; Yong ZHANG

Chinese Journal of Veterinary Science.2004;24(6):599-602.

In present study,comparisons were carried out to develop and improve in vitro mauration(IVM) systems of goat oocytes. Oocytes were cultured in TCM-199 supplemented with(1)10% sera (either estrous goat serum (EGS) or fetal bovine serum(FBS)) + 20 mg/L luteinizing hormone (LH) + 10 mg/L follicle stimulating hormone (FSH) + 1 mg/L estradiol (E2); (2)10% EGS with different gonadotropins(LH: FSH) at a concentration of 5 mg/L: 0.5 mg/L or at 20 mg/L: 10 mg/L with0. 075 IU/mL human menopausal gonadotropin(HMG),1 mg/L estradiol 17β; (3)10% EGS+ 0. 075 mg/L HMG+10-20 μg/EGF. The culture was also performed by M199 supplemented with 10% EGS+0. 075 mg/L HMG+ 10-20 μg/L EGF into ultra-filtrated water which was derived either from self-producing or from purchased for use. The oocytes were cultured at 38 ℃,5% CO2 in air for 24 h,and the meterphase Ⅱ stage oocytes were examined under dissecting microscope. The results showed that EGS was better than FBS in supporting goat oocyte IVM. An addition of HMG in M199 could improve oocyte maturation and induce a higher percentage of metaphase Ⅱ oocytes compared to gonadotropins. 10-20 μg/L EGF improved goat oocyte maturation but the influence was not significant. Fresh,high quality water was vital for oocyte IVM. In conclusion,under our conditions with IVM ,the best result in maturation of goat oocyte has been M199 supplemented with 10% EGS+0.075 IU/mL HMG+10-20 μg/L EGF and prepared in fresh purified water.

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Effect of Zinc on Peripheral Blood T-lymphocyte in the Duckling

Hengmin CUI ; Jing FANG ; Xi PENG ; Junliang DENG

Chinese Journal of Veterinary Science.2004;24(1):66-68.

150-day-old Tianfu ducklings were divided into three groups,and fed with diets as follows :Zn deficient (22. 9 mgZn per kg diet),controls(100 mg Zn per kg diet) and Zn toxic (1 300 mg Zn per kg diet) for seven weeks (Zn deficiency,ZD)or four weeks (Zn toxieity,ZT). The ANAE+ positive ratios of the peripheral blood T-lymphocytes were much lower (P＜0.01 ) in Zn deficient and toxic groups than in the control group. The results showed that Zn deficiency and toxicity would suppress the development of T-lymphocytes and reduce the peripheral blood T-lymphocyte populations. Potential mechanisms underlying these observations are also discussed.

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Polymerase Chain Reaction Detection for Porcine Endogenous Retrovirus in 4 Pig Cell Lines

Maomin LU ; Hong JIN ; Ruichun DENG ; Jianguo WANG ; Shu YANG ; Jingfeng XIONG ; Zhuang DING ; Jingang ZHANG

Chinese Journal of Veterinary Science.2003;23(1):1-3.

Porcine endogenous retroviruses (PERV) can infect human cell in vitro, which raised widely concerns re-garding the transmission of PERV to xenograft recipients. It's essential to establish a method for detection of PERV.3 pairs of primers were synthesized according to the sequence of gag, pol and env gene of PERV. Polymerase chainreaction (PCR) and reverse transcription PCR (RT-PCR) assays were performed for detection of PERV provirusDNA and PERV specific mRNA. The results showed that provirus DNA and mRNA of PERV existed and expressedin all 4 tested cell lines. The sizes of amplified fragments are identical with the predicted. These methods may be suit-able for monitoring PERV in other cells or tissue.

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Halothane Gene and Its Effect on Meat Produc tivity in Suzhong Line 1 and 2 Pigs

Xiaobo XU ; Tiezheng LIU ; Kangpu XU ; Yunshan GE

Chinese Journal of Veterinary Science.2000;20(4):397-400.

Three hundred and one piglets of Suzh ong line 1 and 2 were chosen for halothane test(HT), with the halothane positive rate of 1.33%. 34 halothane negative(HN) piglets were randomly selected for gen otype testing and 6 of them were found to be heterozygous. The results showed th at genotype frequencies of HalNN, HalNnand Halnn w ere 81.26 %, 17.4% and 1.33%, respectively, while the frequencies of N gene and n gene wer e 89.97% and 10.03%, separately. 4 pigs with genotype Halnn gave worst meat quality appearing 3 typical and 1 slight PSE meat. Pigs with genotype Hal N Nor HalNndid not show PSE meat. 208 offspring from 20 HN sows mat ed wit h halothane positive(HP) boars, 21 piglets from 4 litters were HP, with low dail y gain, low feed efficiency and bad meat quality but high dressing percentage, l arge loin-eye area and high lean meat percentage.

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Construction and screening of a cDNA expresssion library of Clonorchis sinensis adult worm

Xiuping WU ; Liqang GAO ; Ling ZHANG ; Hongkuan DENG ; Xiangye LIU ; Chunyan YE ; Zijian WANG ; Xuelin WANG ; Feng WANG ; Mingyuan LIU

Chinese Journal of Veterinary Science.2009;29(7):868-872.

In order to screen antigenic genes of Clonorchis sinensis (C.sinensis).Firstly,a cDNA expression library from adult worms of C.sinensis was successfully constructed with lambda ZAP express vector.Total RNA of C.sinensis adult worms were extracted by the Trizol reagent and mRNA were further purified through oligo-dT cellulose.The first strand eDNA was synthesized by using MMLV reverse transcriptase.After the synthesis of the second strand,the cDNA were purified by CHROMA SPIN-400 kit and then ligated with lambda ZAP express vector,then packaged in vitro and amplified.The original library contained 1.5 × 106 pfu cDNA clones,the titer of amplified library reached 1.5 × 1010 pfu/mL,in which about 99% clones were recombinants and most of insert DNA fragments were 0.4-2.0 kb.Secondly,immunoscreened using the naturely infected man serum from C.sinensis.The positive clones were sequenced and analyzed.From 2.0 × 105 recombinant clones of the eDNA library,41 positive clones were obtained.Sequence analysis indicated that the cDNAs encoded proteins including glycine rich antigen 2,proline rich antigen 2 and antigen Cs44 from C.sinensis,anothers were lower similarity to predicted protein from Nematostella vectensis,transcription elongation factor GreA from Bartonella quintana str.Toulouse and NM_132090 CG3446 gene product in Drosophila melanogaster from Schistosoma japonicum.These data may form a foundation for identifying recombinant antigens that can be used in the diagnosis or vaccination against clonorchiasis.

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Cloning and expression of the σC gene of avian reovirus

Xiaobo HUANG ; Shanshan LING ; Sanjie CAO ; Xintian WEN ; Cunwei WANG ; Yuankun CHEN

Chinese Journal of Veterinary Science.2009;29(7):836-840.

The σC gene of ARV S1133 was designed to amplify by reverse transcription chain reaction(RTPCR).The σC gene was inserted into the vector pMD19-T,identified by PCR method and restriction enzyme,and sequenced.It showed that the insert cloned gene fragment was the σC gene of ARV.Then the gene was inserted intc the pET32a(+) and indicated that fusion expression vector pET32a-σC was constructed.The recombinant fusion protein was highly expressed in E.coli BL21 induced by 1.0 mmol/L IPTG for 5 hours in the form of inclusion bodies.The weight of recombinant fusion protein molecular is 54 000.Western-blot with ARV antibodies against the fusion protein showed the recombinant protein has a favourable reactivity.

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Effects on humoral immune response to classical swine fever vaccine after experimental infection of porcine circovirus type 2

Xingkui SI ; Xin GUO ; Hanchun YANG

Chinese Journal of Veterinary Science.2009;29(7):826-829.

The levels of the antibody response against,classic swine fever (CSF) vaccine were measured by blocking ELISA at different time for pigs vaccinated with CSF vaccine (CSFV group,n= 3),pigs inoculated with porcine eircovirus type 2 (PCV2) and with CSF vaccine two weeks later (when PCV2 viremia was detected by PCR,PCV2/CSFV group,n=3),or inoculated with both CSF vaccine and PCV2 at the same time (CSFV/PCV2 group,n=3),respectively.And distribution of genome or antibodies specific to PCV2 of the PCV2 infection group,PCV2/CSFV group and CSFV/PCV2 group were also detected by indirect ELISA or PCR,respectively.The results showed that experimental infection of PCV2 was successful.In pigs inoculated with both PCV2 and CSF vaccine,the antibody response to CSF vaccine was significantly lower than that of animals treated with CSF vaccine vaccination alone at 52 days postinoculation (DPI).The average titers of antibodies against CSFV in animals of CSFV group were obviously higher than those of the PCV2/CSFV or CSFV/PCV2 group at 49 and 52DPI,respectively.The positive rate of antibodies against CSFV in pigs of CSFV group was 100% in comparison with that of animals inoculated with PCV2 and the vaccine (only 67%).The results suggested that the infection of PCV2 could suppress the antibody response to classical swine fever vaccine.

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