Laboratory Animal Science

Establishment of IFA in Detecting Japanese Encephalitis Virus

WANG Ji,GAO Zheng-qin,WEI Li,FU Rui,YUE Bing-fei,HE Zheng-ming

2010, 27(04): 1-5.

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Objective Establishing the method of detecting the Japanese encephalitis virus(JEV) in swine and the biological materials from the swine with Immunofluorescence assay(IFA).Methods Selected JEV sensitive cell,titrate the virus’TCID50 and prepare the antigen slides according to GB IFA.titrate the best working density of immune fluorescence and carry out the experiments of specificity,sensitivity and stability.Result Selected BHK21 cell as JEV sensitive cell,the virus’ TCID50 is 10-8.9.mL-1;The best working density of immune fluorescence is 1∶ 200;There is no cross-reactivity with Classical swine fever virus(CSFV) and Porcine parvovirus(PPV),the experiments of sensitivity and stability show that the detection sensitivity is 1∶ 2 560 in different time;The lowest detected virus titer is 10-6.5.mL-1.Conclusion The IFA method is good in sensitivity,specificity and stability,as a result of which IFA can be used in detecting the swine and the biological materials from the swine.

Expression of the Hemagglutinin and Neuramidinase Gene of Influenza A Virus H3N2 in P. methanolica

LI Zi1,ZHANG Ye1,TANG Qi-hui2,CHEN Yu-bao3,YU Zai-jiang1,XIN Li1,CHEN Yong-kun1,CHEN Qing-xuan2,SHU Yue-long1

2010, 27(04): 6-10.

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Objective To clone,express and characterize the HA(hemagglutinin)and NA(neuramidinase,NA) protein of influenza virus H3N2.Methods On the basis of successful clone the full length HA and NA gene and sequence analysis of influenza virus H1N1,and the genes were ligated into pMET A.An expression vector pMET A / HA(52 bp-1 549 bp),pMET A /NA(121 bp-1 260 bp)were constructed and expressed in pMAD16 induced by methanol.Recombinant protein was purified through Ni2 + affinity chromatography column.Western Blotting and ELISA were used to determine the antigenic of the recombinant protein.Results The recombinant capsid gene can be overexpressed in P.methanolica.SDS-PAGE showed that the product is a dimer.The purity of the protein is greater than 95%.ELISA and Western Blotting showed that the recombinant protein has good antigenic.Conclusion the HA and NA Protein of influenza virus H3N2 has been successful cloned and expressed,which could be useful for developing diagnose reagents or vaccine of H3N2.

Study on Genetic Monitoring of Frozen Embryos for C57BL/6J Mice

LIU Xian-ju,HUANG Xin,WANG Yan-rong,LIU Yun-bo

2010, 27(04): 11-14.

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Objective To analysis and study if change on genetic plasma of four F1 C57BL /6J.Method By both the methods of inbred mice genetic monitoring(GB /T 14927.1-2008) and inbred strains by analysis of microsatellite polymorphisms,to monitor the genetic plasma of four F1 offspring from vitrification-thawed embryos.Result The genetic plasma non-changed by frozen embryos for C57BL /6J.Conclusion These results suggest the method of the offspring from vitrification-thawed embryos(EFS40,straw) non-damaged the genetic plasma for frozen embryos,and the genetic characteristic is stable.So it’s an advisable method for frozen embryos of mice.

Construction and Identification of Mouse Bpi Conditional Gene Knockout Vector

KANG Yun1,2,LIN Fu-yu2,TENG Yan2,HOU Ning2,CHENG Xuan2,Lü Zhe1,KONG Qing-li1,AN Yun-qing1

2010, 27(04): 15-20.

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Objective To construct Bpi conditional gene knockout vector will lay the foundation of preparing the Bpi conditional knockout mouse for further Bpi gene functional study.Methods Using Cre /LoxP system,two LoxP sequences were inserted into intron 1 and 3 to flox exon 2 and 3 of Bpi.The 3.1 kb Bpi genomic DNA fragment containing exon 2 and 3 used as upstream homologous arm and middle homologous arm and the 4.9 kb Bpi genomic DNA fragment used as downstream homologous arm were obtained separately by LA-PCR using mouse ES cells genomic DNA as template.The recombinant plasmid of pBSKⅡ-5SLoxP and ploxPFRTNeo-3L were then used to obtain Bpi conditional gene targeting vector by sub-cloning.Results The recombinant plasmid and final conditional targeting vector were confirmed by restriction enzyme digestion and sequencing analysis.Conclusion A conditional gene targeting vector of mouse Bpi has been successfully constructed.

Analysis Genetic Stability on Production Expansion Colony of Inbred Strain A2a Knockout Mice by Sequencing of mtDNA D-loop

WANG Jin-dan1,GUAN Min-qiang2,Li Yuan2,CHEN Xi-wen1,JIN Long-jin2

2010, 27(04): 21-23.

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Objective To analyse genetic polymorphisms and genetic stability in mtDNA D-loop sequence on the fifth generation of A2a mice.Methods Fifty heterozygote mice were selected and reproduced to five generations.mtDNA D-loop region of them were amplified respectively by PCR with two pairs of primers and then sequenced.The result of DNA sequence compared with standard sequence of mice.Result The Sequences of D-loop gene of the Fifty A2a mice ware the same totally,and were the same as the standard sequences of mice.Conclusion The A2a knockout mice colony showed good genetic stability and provide the invaluable resource for experimental animal research as well.

Preliminary Studies for Genetic Homozygosity of SX1 F_(22th) Inbred Strain Mice

WEN Jian-hua1,LI Jian-guo1,TIAN Huan-cheng1,ZHANG Wei,QIN Xiu-jun,REN Lian-sheng2

2010, 27(04): 24-26.

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Objective Preliminary identifying the 22th SX1Inbred Strain Mice’s genetic homozygosity of Shanxi Tumor Institute.Methods The study preliminary identified 22th Kunming Inbred Strain Mice ’s genetic homozygosity of Shanxi Tumor Institute by using for biochemical marker methods of genetic quality examination and skin grafting test.Results Thirteen biochemical markers of six inbred strain mice examining dont find heterogeneous gene;ten inbred strain mice exanming don’t find acutely rejecting.Conclution The study may preliminary judge that the 22th Kunming Inbred Strain Mice of Shanxi Tumor Institute self-fostering is in line with the requirement of laboratory animal genetic quality examination.

The Intervention Effect of Hu Qi San on Mice Alcoholic Liver Disease

XUE Xiao-wei,FENG Ping,DONG Kun,ZHAO Na,WANG Xue-jiang

2010, 27(04): 27-32.

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To observe the different effects of wine spirit and red wine on liver;to interpret the relationship between alcoholic liver disease and hepatoma in terms of oxidative stress and energy metabolism then to explore pharmacology mechanism of Hu Qi San(HQS) intervening the malignant transformation of alcoholic liver disease.KM strain male mice were randomly divided into normal group,white spirit group,red wine group and HQS group.After 14 days,the MDA,SOD and CAT in serum were detected by colorimetry.HE staining,PAS staining,G-6-P staining,SDH staining and ATPase staining were on the liver tissue.In white spirit group,the contents of MDA,the index reflected lipid peroxidation,were significantly increased,the activities of SOD,CAT and the key enzymes about energy metabolism such as G-6-P,SDH and ATPase were restrained.The red wine had no damage on liver.HQS decreased the level of MDA,improved the activities of antioxidant enzymes and the energy metabolism key enzymes;PAS staining revealed an increase of glycogen granules.These experimental results indicated that long-term intake of ethanol causes some damage to liver,but red wine causes no.HQS has intervention effect on alcoholic liver disease.

The Experimental Study of Qiangxinkeli on Activating Blood Circulation to Dissipate Blood Stasis Effects

QIN Wen-Yan,DONG Bo,CHEN He,QI Yue,MENG Li,ZHAO Jin-ming

2010, 27(04): 33-36.

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Objective To study the activating blood circulation to dissipate blood stasis effects of Qiangxinkeli(QXKL).Methods Established the mesentery microcirculation disturbance with adrenaline in mice to observe the effect of QXKL on the mesentery microcirculation disturbance;subcutaneous injecting adrenalin hydrochloride twice,during the period,stimulating the rats with the ice water to induce the acute blood stasis model to survey the influence on the hemorheology.Results QXKL low dose 21.5 g /kg、middle dose 43 g /kg and high dose 86 g /kg could accelerate the blood flow rate,improve the blood flow state,thereby ameliorate the microcirculation disturbance in mice;QXKL middle dose 30 g /kg and high dose 60 g /kg could reduced the blood viscosity in high、 midst and low shear rate in model rats remarkably,the high dose could degrade the hematocrit and the midst dose could accelerate the erythrocyte electrophoresis time.Conclusion QXKL possesses the obvious activating blood circulation to dissipate blood stasis effects.

Protective Effects of the Naoshuantong Granula on Experimental Cerebral Ischemia

MENG Li,JIAO Fu-ying,QIAO Min,XIANG Shao-jie,QIAO ju-jiu,ZHANG Ying,QIN Wen-yan

2010, 27(04): 37-39.

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Objective To study the protective effect of Naoshuantong granula(NSTG) on experimental cerebral ischemia in rats.Methods The impacts of ordinary pressure hypoxia were observed in mice after being given NSTG with the methods of mouth breathing after decapitation;anti-hypoxia effect were investigated by normobaric hypoxia;Evans blue contents were measured to evaluate the blood-brain permeability effects by injection of evans blue into mouse vein;cerebral ischemia reperfusion injury in rats were studied by ligating bilateral common carotid artery.Results NSTG could significantly prolong gasping time after decapitation mice;NSTG could not extend the survival time trend under hypoxia at normal pressure;NSTG could increase brain evans blue content and the blood-brain permeability in mice with the middle dose or high dose;decrease water content in brain tissue and improve edematous state after ischemia-reperfusion injury.Conclusions NSTG possesses the obvious effect on experimental cerebral ischemia.

The Research on Anti-tussive and Phlegm-removing Effects of Guben Qingfei Tiegao

LI Xian-hua1,SONG Da-fu1,GAN Yu1,DU Jia-lin1,GU Xiao-ying2,AN Ran2

2010, 27(04): 40-41.

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Objective To survey the anti-tussive and phlegm-removing effects of Guben Qingfei Tiegao.Methods apply the spraying concentrated ammonia method on mice to observe the anti-tussive effects of Guben Qingfei Tiegao;phenol red secretion test is used to observe the phlegm-removing effects of Guben Qingfei Tiegao in mice.Results Guben Qingfei Tiegao could prolongate the latent period of cough obviously,decrease the frequency of cough,increase the amount of phenol red secreted from mice's bronchus.Conclusion Guben Qingfei Tiegao has the effects of anti-tussive and phlegm-removing.

Effects of Transfer Factor (TF) on Blood Physiological and Biochemical Parameters of Infant Macaca mulatta

YU Wen-hai1,HE Zhan-long1,HUANG Fen2,CHEN Li-xiong1,LU Shuai-yao1,ZHAO Yuan1,WANG Jun-bin1,YANG Jing-kai1,YANG Liang-yu3

2010, 27(04): 42-45.

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Objective The studies were performed on the infant Macaca mulatta that injected with Humans’ transfer factor to analyze the effects after injection.It will provide a reliable and scientific data to develop and prepare a monkey-specific medicine.Methods The physiological and biochemical change of five infant Macaca mulatta(5 ♂,5♀) were measured by automatic blood counting instrument and automatic biochemical analyzer,respectively.Results There were most significant differences in MCH(P < 0.01) and significant differences in MCHC(P < 0.05) compared with control group;in the three experimental groups,MCH showed the most significant differences(P < 0.01),and WBC,PCT,Mid #,RDW,PLT,PDW showed significant difference(P < 0.05).The biochemical parameters of blood were most significant differences in TG,UA,AST,AKP,LDH(P < 0.01) and significant differences in ALT(P < 0.05) compared with control group;compared among three experimental groups,there were significant differences in LDH,AKP,AST(P < 0.05),and no significant differences in other biochemical parameters.Conclusion There were obvious effects on some blood physiological and biochemical parameters in infant Macaca mulatta after injected with transfer factor,which prepared for human beings.The results provided effective data to utilize and produce medicines for animals.

Infectious Investigation of Lymphocytic Choriomeningitis Virus(LCMV) in Laboratory Animal Rat

TONG Wei,QIAO Hong-wei,WEI Qiang,ZHANG Li-fang,DAI Xiao-wei,LIU Xian-ju,WANG Yan-rong,LIU Yun-bo

2010, 27(04): 46-48.

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Objective To investigate the infection status of LCMV in laboratory animal rat,we inspected LCMV antibody of rat serum samples preserved in our laboratory.Methods Immunofluorescent(IFA) from the current national standard was used in testing LCMV antibody of 172 rat serum samples from 48 non-quality controled animal,100 clean animal and 24 specific pathogen free animal respectively.Results 15 samples in non-quality controlled rat were tested as LCMV antibody positive(positive rate 33.25%),and the highest antibody 滴度 is 1∶ 1280;The samples of SPF and clean animal were tested as LCMV antibody negative.Conclusions The rat may be infected by LCMV,so we suggested that the LCMV should be added into microbiological quality control standard of rat as a monitoring item.

The Reconstruction of Animal Experiment Facility for Barrier System

LU Xuan-cheng1,LI Xiao-yan1,ZHANG Qi2,LI Jun-yan2

2010, 27(04): 49-54.

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In order to make the animal experiment facility to adapt to the developing life science,the original animal experiments facilities could be reconstructed.The following items should be pay more attention to,including standardized personnel,laboratory animals and air mobile transmission direction,the Automatic control of temperature,humidity and pressure gradient,facilities fire safety,increased communication,monitoring,access control and music systems.The new barrier system could meet the requirment of national standard and scientific needs.

Safety Reevaluation of Tiopronin Injection

HONG Hua1,ZHAO Xiang-ren2,LI Zhi-man1

2010, 27(04): 55-58.

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Objective Reevaluate the safety of soled tiopronin injection according to the feedback of its existing certain clinical reaction.Method Make an experiment of tiopronin injection on vascular stimulation,hemolysis,and sensitization.The dosage conversion between people and animals is 1∶ 20(rabbits and guinea pigs).Results Tiopronin injection has no obvious stimulatory effect on the marginal ear veins of rabbits,no obvious influence of hemolyzation and agglutination on rabbit red blood cell,and no allergic action on guinea pigs.Conclusion The safety of soled tiopronin injection used as the clinical intravenous injection is good.

Exploration of Establishing ⅡDiabetes Mellitus Rats Model by Complex Methods

DONG Jian-yi,WANG Liang,WANG Fu-jin,LI Hui-ling,ZHAN Hong-wei,WANG Jing-yu

2010, 27(04): 59-61.

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Objective To observe the effect of stability and tolerance of the Ⅱdiabetes mellitus SD rats model.Methods SD rats are injected intraperitoneally with 25 mg /kg streptozotocin once after fed high fat diet 4 weeks.Results when the SD rats were injected STZ one week,the blood glucose of SD rats were stably higher then 6.78 mmol /L.During the 22 days observation,Although the blood glucose values of SD rats have fluctuated at a latter stage,the stability and tolerated of blood glucose of SD rats were well,were over 6.78 mmol /L.Conclusion The Ⅱdiabetes rats model can be established successfully with feeding high fat diet and injecting a low dose STZ(25 mg/kg) once.This method is simple and use a small amount of medication,the model has hatter stability and tolerance.

An Approach to Biosafety Containment of Animal Laboratory

HAO Zhi-hui,ZHAO Hou-de

2010, 27(04): 62-64.

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Animal studies have been applying in all fields of life science,but they bring some biohazards to workers,which may result from animal aerosol,zoonosis and laboratory infection.We must pay more attention to the containment of laboratory biosafety.We can improve our consciousness of self-protection and containment skill through serial training.Furthermore,we must establish standard operation procedure(SOP) and policy for administration and supervision on containment of biosafety in order to make sure people avoid from the laboratory infection.

Bulletin Board of Experimental Animal Resource Sharing Network Design and Thinking

LI Zhi-man1,TONG Xiao-hua1,HONG-Hua1,DONG Yu-jie2,LI Qing2

2010, 27(04): 65-67.

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In order to realize the experimental animals of information resources sharing network,we design and construction of the experimental animal resource sharing information network,the bulletin board to park the production unit can supply varieties of experimental animals and through the network to electronic form,for the display mode of experimental animals use units to provide fast,reliable,also for flying inquires the manager 's decision to provide reference data,local resources,so as to make full use of various participation and sharing.

Connected to Aseptic Isolator Respectively and Ultra Clean Bench,and Laminar Flow Hood

LI Zhi-man1,TIAN Zhi-ning,ZHANG Xiao-hong,TONG Xiao-hua

2010, 27(04): 68-69.

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In order to solve the sterile isolator in open environment clean level SPF animal experiments,the question,we will with sterile isolator worktable,anti-oil laminar covers respectively,straight after modification of the dock barrier facilities,low cost,simple and safe operation,to improve work efficiency,the experimental effect is good.

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