奶牛乳铁蛋白基因部分序列的PCR—SSCP分析

本研究采用PCR－SSCP方法对奶牛乳铁蛋白基因的第7、12外显子和5′调控区部分序列进行多态性分析，发现该基因5′调控区一段长227bp的DNA片段上存在多态性。经过对突变纯合（BB）的两头奶牛个体进行测序分析后证明，位于起始位点上游CAAT框－926和－915位置分别具有G→A及T→G点突变，同时在－839和－811位置分别具有C和T单碱基的。等位基因A、B的频率分别为0.898和0.102。     

甘蓝Ogura胞质雄性不育基因的RAPD标记筛选*

有关利用RAPD[1]技术进行分子标记的研究报道很多,如王俊霞等[1]对甘蓝型油菜Pel CMS育性恢复基因和王晓武等[2]对甘蓝的一个显性核雄性不育基因均进行了RAPD标记.我们经过多年的选育,已经成功把外源胞质雄性不育基因转育到甘蓝自交不亲和系上,现已成功选育出不育性稳定和经济性状优良的甘蓝雄性不育系.为了更深入的研究其不育机理,利用RAPD分子标记技术对其不育基因进行了标记,为以后开展甘蓝杂优育种提供标记基因打下基础.     

Research Progress on Function of Pig Bactericidal/Permeability-increasing Protein (BPI) and Its Application in Resistance Breeding

Bactericidal/permeability-increasing protein (BPI) has a strong effect on sterilization (mainly for G^- bacteria),neutralizing the activity of lipopolysaccharide (LPS) and enhancing the phagocytosis of mononuclear cells and neutrophils to pathogenic bacteria.The biological functions of BPI have been researched widely in recent years,which is known as "super antibiotic" and has been explored by many scholars as a candidate gene for resistance.This author summarized the research progress and application prospect of the BPI gene in the pig resistant breeding by introducing the structure,biological function of BPI gene and its relationship with the resistance,which was aimed to provide theoretical references and basis for the function research of pig BPI gene and its practical application in resistance breeding in future.     

Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.     

Study on Expression Difference of IGFBP-5 Gene in Different Tissues of Kazakh and Yanqi Horses

The study aimed to explore the mRNA expression pattern of insulin-like growth factor binding protein-5 (IGFBP-5) gene in different tissues of Kazakh and Yanqi horses.The expression of IGFBP-5 gene in different tissues of heart,liver,spleen,lung,kidney,small intestine,large intestine,cecum,intercostal muscles,longissimus dorsi muscles,brachialis muscle and gluteus in two horses were detected by Real-time quantitative PCR and compared the mRNA expression in the same tissues of two breeds.The results showed that the expression of IGFBP-5 in longissimus dorsi muscle and brachialis muscle of two breeds were significantly higher than other tissues including heart,liver,spleen,lung,kidney,small intestine,large intestine and cecum (P<0.05),and was the lowest in large intestine.The expression of IGFBP-5 in kidney,small intestine,large intestine,cecum,longissimus dorsi muscle and brachialis muscles of Yanqi horse were higher than in the same part of Kazakh horse,and among those in longissimus dorsi muscle and large intestine of Yanqi horse were extremely significantly higher than in the same part of Kazakh horse (P<0.01),and in small intestine and cecum of Yanqi horse were significantly higher than in the same part of Kazakh horse (P<0.05).The test was for further researching the biological function of IGFBP-5 gene,and it could provide a theoretical basis for genetic improvement of production performance of horse in our country.     

Cloning and Bioinformatic Analysis of L1 Gene of BPV from Guizhou Province

In order to study and analyze L1 gene of bovine papillomavirus（BPV）in Guizhou province,the L1 gene of BPV-GZ01 strain was amplified,cloned and sequenced using bioinformatic softwares and methods,and the secondary structure,tertiary structure,B-cell preponderant epitope,conserved domains analysis, transmembrane domain and signal peptide of L1 gene were predicted.The results showed that the length of L1 gene was 1 494 bp,encoding 497 amino acids.The L1 gene of BPV-GZ01 strain shared an amino acid identities of 98.6%,99.4%,98.4%,94.4% and 91.3%,and a nucleotide identities of 99.1%,99.8%,99.4%,87.6% and 82.8% with those of BPV2,BPV2-SW01,BPV2-AKS01,BPV13 and BPV1 strains,respectively.The results of phylogenetic tree analysis indicated that there was a close relationship between BPV-GZ01 and BPV2-SW01 strains.The prediction of secondary structure of L1 protein indicated that the random coil,extended strand and alphahelix took a higher percentage.The L1 protein was supposed contain 6 potential antigen epitopes.And no transmembrane domains and no signal peptide were found.The tertiary structure of L1 protein was curved spiral structure.These results provided a theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of BPV.     

The Fluorescent Characterization and Analysis of Two Brucella Attenuated Vaccine Infecting Mouse Macrophagocyte

The experiment was conducted to discuss the difference of binding time of green fluorescent protein B.melitensis M5 (GFP-M5) and B.abortus S19 (GFP-S19) infecting the mouse macrophagocyte (RAW264.7),lysosome,endoplasmic reticulum and golgi body in the initial stage and compare the binding rate of GFP-M5,GFP-S19 with organelle in different timeline,respectively,by confocal laser scanning microscope (CLSM) and flow cytometry.The result showed that GFP-M5 and GFP-S19 were successfully constructed.The intracellular survival ability of Brucella M5,Brucella S19,GFP-M5 and GFP-S19 were not obvisouly affected after infecting RAW264.7.GFP-M5 and GFP-S19 could enter the macrophagocyte in 30 mins,and in 2 h the Brucella could reach lysosome,endoplasmic reticulum and golgi body.In addition,the binding time for two attenuated vaccine did not show differences in 1,2,3 and 4 h.The content of GFP⁺ cell produced by RAW264.7 infected by GFP-M5 and GFP-S19 did not show significant differences (P>0.05).Therefore,the two strains did not have significant differences in the invasion ability in the initial stage of infecting host cell.     

Effect of Se and VE Supplement on Semen Quality,Antioxidant Enzyme Activities and Heat Shock Protein Expression of Goat in High Temperature Season

This experiment was conducted to study the effect of selenium and vitamin E supplement on semen quality, antioxidant enzyme activities and heat shock protein expression of goat in Hainan high temperature season.16 adult Hainan Black goat with good health and approximate weight were randomly divided into 4 groups, fed with basal diet(control group), basal diet+0.5 mg/kg Se(Se group), basal diet+100 mg/kg VE(VE group), and basal diet+0.5 mg/kg Se+100 mg/kg VE(Se+VE group), respectively.The experimental period was 93 d.Semen samples were collected in the last week of the experiment on two consecutive days.The semen quality, antioxidant enzyme activities and heat shock protein expression were analyzed.The results showed that compared with control group, the ejaculate volume was not significantly affected by Se or VE supplement(P>0.05).Sperm density and sperm motility were increased significantly by Se and VE supplement(P<0.05), and the abnormal rate was decreased extremely significantly(P<0.01).The goats fed with Se and VE also had higher activities of GSH-Px(P<0.01), SOD(P<0.05), CAT(P<0.05) and T-AOC(P<0.01), and lower MDA concentration in seminal plasma(P<0.05).The relative expression levels of HSP70 and HSP90 mRNA in supplement groups were decreased extremely significantly(P<0.01).However, there were some certain differences between the Se and VE supplement groups on semen quality and heat shock protein expression.In conclusion, the supplementation of Se and VE could help to improve goat semen quality by increasing the sperm density, sperm motility, the antioxidant enzyme activities, and decreasing the abnormal rate in hot season of Hainan.Finally, Se and VE supplement had good effects on relieving the environment heat stress.     

Protective Effect of Sulforaphane on Reproductive Function of Male Mice with Cadmium Poisoning

The study was aimed to explore the protective effect of sulforaphane (SFN) on the reproductive function of male mice with cadmium poisoning.40 healthy clean grade male Kunming mice were randomly divided into four groups:control group (H₂O),cadmium chloride group (2.3 mg/kg CdCl₂),sulforaphane group (10 mg/kg SFN),sulforaphane + cadmium chloride group (10 mg/kg SFN+2.3 mg/kg CdCl₂),and continuous administration for 10 d,all mice were executed by dislocated cervical vertebra at 2 d after the last administration,and then the pathologic changes of testicular tissues,organ coefficient of testicle and epididymis,sperm quality and concentration of testosterone were tested.Additionally,the contents of GSH and MDA,and the activities of T-SOD in testis were also detected at the same time. Compared with the control group,pathology damages were observed in cadmium chloride group,organ coefficient of testis and epididymis,sperm quality and levels of testosterone extremely significantly decreased (P<0.01),the activities of T-SOD and GSH content were extremely significantly decreased (P<0.01),and the concentration of MDA was extremely significantly enhanced (P<0.01).Compared with the control group,the activity of T-SOD and concentration of GSH in sulforaphane group were significantly increased (P<0.05),and the concentration of MDA was not significant different between the control group and sulforaphane group (P>0.05).While compared with the cadmium chloride group,the sperm motility rate and sperm total count in sulforaphane and cadmium chloride group were extremely significantly increased (P<0.01),the organ coefficient of testicle and epididymis was increased significantly (P<0.05),the concentration of GSH and activity of T-SOD in testicular tissue were extremely significantly increased (P<0.01),and the concentration of MDA was extremely significantly decreased (P<0.01).The results indicated that sulforaphane had the protection effect on reproduction function of male mice with cadmium poisoning.     

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.