不同培养液对体外培养的小鼠胸腺上皮细胞增殖的影响

分离纯化小鼠胸腺上皮细胞（mTEC）,以DMEM、DMEM/F12、RPMI 1640 3种基础培养液加10%胎牛血清完全培养液进行培养, MTT法和流式细胞术分别检测mTEC的增殖及细胞周期,筛选出最适合mTEC生长的培养液。结果表明,DMEM培养的mTEC增殖快,细胞增殖指数明显高于DMEM/F12、RPMI 1640培养液,表明DMEM是mTEC体外培养的最佳培养液。     

Study on Proliferation and Apoptosis of EBL Cells Induced by P48 Protein of Mycoplasma bovis

The aim of this study was to investigate the effect of P48 protein on proliferation and apoptosis of embryonic bovine lung (EBL) cells.In this study,samples which co-incubated with P48 protein and EBL cells in different concentrations at different time points were collected,the proliferation rate of the cells was detected by MTT method,and the changes in the nuclear morphology of EBL cells were observed by DAPI staining method.Meanwhile,flow cytometry was used to detect the apoptosis rate of EBL cells induced by P48 protein.Real-time quantitative PCR was used to detect the changes in mRNA level of apoptotic marker genes,and Western blotting tested the Bax and Beclin-1 protein expressions.The results showed that under the condition of 72 h and 10 μg/mL of protein concentration treatment,P48 extremely significantly inhibited EBL cells proliferation (P<0.01),while 0.1 and 0.5 μg/mL protein concentration had no inhibitory effect (P>0.05).The nuclear morphology showed no significant change after protein induction for 12 h,but wrinkled and condensed at 24 h.The nucleus was fragmented,and a sprouted apoptotic body was appeared at 48 and 72 h.Apoptosis related genes expression showed no obvious increase at 2 and 12 h at mRNA level,but gradually increased at 24,48 and 72 h,and it showed a time-dependent manner.Accordingly,the expression of apoptosis marker proteins Bax and Beclin-1 significantly increased.Flow cytometry analysis showed that the apoptosis rate of EBL cells induced by P48 protein was 48.44%.In conclusion,P48 recombinant protein of Mycoplasmas bovis inhibited the proliferation of EBL cells and promoted their apoptosis,which provided reference for revealing the pathogenic mechanism of Mycoplasmas bovis.     

Seroprevalence of Antibodies to Chlamydophila psittaci in Zoo Workers in Brazil

To evaluate the prevalence of antibodies to Chlamydophila psittaci 364 serum samples were collected from veterinarians, biologists, animal scientists, veterinary students, animal keepers and others employees in 20 zoos, and from veterinary practitioners in 10 Brazilian states. Subjects ranged from 15 to 64 years of age, with 268 (74%) males and 96 (26%) females. Chlamydial antibodies were determined by the complement fixation test (CFT) and specific anti‐C. psittaci IgG antibodies were determined by the microimmunoflurescence (MIF) test. Complement fixation test showed 23.9% (87/364) and MIF test showed 4.7% (17/364) positive serum samples. Titres ranged from 16 to 256 in both assays, demonstrating evidence of recent or current infection. Although chlamydial antibodies were detected in workers of seventeen zoos, MIF test only detected specific C. psittaci antibodies in seven of them. Previous psittacosis infection was suspected in eight workers of two zoos, five of whom reported having pneumonia, while employed at the zoos. However, diagnosis was not established in any of these cases in the past. Results indicated the occurrence of infection and previous contact of Brazilian zoo workers with C. psittaci, as well as the zoonotic potential of psittacosis in this risk population. Other studies are necessary to evaluate the risk factors of infection in this population. This seroepidemiological survey confirmed the need to adopt preventive measures to control avian chlamydiosis and protect the health of zoo workers in the country.     

自身启动子控制的卡那霉素抗性基因在哺乳动物细胞中表达的检测

为了研究卡那霉素抗性(KanR)基因能否在哺乳动物细胞中表达以及用含相同抗性基因重组质粒防治奶牛乳腺炎的安全性,用PCR从重组质粒p215C3LYZ中扩增得KanR基因,将其克隆入原核表达载体pQE-31,用含卡那霉素(Kan)琼脂平板筛选得KanR重组菌,经IPTG诱导成功表达了预期大小的Kan抗性融合蛋白;用纯化的重组蛋白免疫小鼠,获得了Kan抗性蛋白抗血清,经Western blotting证明免疫血清特异性良好;分别用重组质粒pQE-Kan和p215C3LYZ转染COS-1细胞,在不含抗生素培养液中培养后分别收集细胞上清和细胞裂解物;将转染细胞上清分为添加和不加Kan组,接种Kan敏感菌DH5α大肠杆菌,培养物的OD600检测结果显示,添加组的指示菌生长被抑制,转染细胞上清中无Kan抗性蛋白表达;以Kan抗性蛋白免疫血清进行的Western blotting结果显示,转染细胞内无Kan抗性蛋白表达;将p215C3LYZ注射奶牛乳腺,用脱脂和浓缩奶样进行Western blotting检测,结果显示试验牛乳中也无Kan抗性蛋白表达。这些试验结果提示,来源于原核大肠杆菌的KanR基因启动子在动物细胞中无转录活性,乳腺注射含KanR基因的重组质粒不会表达对奶牛和人体有害的Kan抗性蛋白。     

H5N1亚型禽流感病毒A/Guangxi/1/2005株抗药机制的研究

为研究H5N1亚型禽流感病毒(AIV)的抗药机制,本研究选取Clade2.3.4亚群中一株对金刚烷胺敏感的人源AIV A/Guangxi/1/2005(H5N1)(S-GX05),用抗流感病毒药物金刚烷胺对其进行定向诱导,筛选出一株抗药性病毒株,命名为R-A/Guangxi/1/2005(R-GX05)。通过全基因测序并与S-GX05全基因序列进行对比,结果显示S-GX05只在其M2蛋白中有一个氨基酸位点发生突变,即A30P;抗药性鉴定这两株病毒的半数药物抑制浓度(IC50)分别为0.9μM和48.9μM,表明R-GX05对金刚烷胺表现出一定程度的抗性。动物实验证实,这两株病毒对BALB/c小鼠的致病性基本一致,均表现出高致病性,其MLD50分别为4.7 log10 EID50和5.0 log10 EID50,两株病毒在小鼠体内各组织脏器中的分布及增殖能力也基本相同。这些结果表明,S-GX05在药物压力下产生抗药性后,并未引起其它生物学特性的改变。A30P的发现为进一步从分子水平上研究H5N1亚型AIV的抗药机制及新型抗流感新药的研发奠定了基础。     

寄生虫细胞因子免疫的研究进展（一）

寄生虫在入侵机体时必须具备逃避机体免疫,破坏宿主免疫屏障才能感染,这种入侵过程必然和宿主机体的免疫系统发生联系,不但使宿主的先天免疫细胞功能缺失,而且获得性免疫也受到抑制。寄生虫感染均不同程度地伴随免疫损伤,宿主在感染寄生虫后产生的免疫应答中,细胞因子作为免疫过程中的介质起到关键作用,主要是调节机体细胞免疫抗虫,保护机体不被感染,其中巨噬细胞是主要功能的承担者。本文将阐述细胞因子在寄生虫感染过程中所发挥的免疫作用,为解决寄生虫感染的防控问题奠定基础,同时为寄生虫的研究提供新的思路。     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

H9亚型禽流感病毒感染引起MDCK细胞内抗氧化功能的变化

将传代MDCK细胞以8×10⁴/cm²的密度接种于6孔细胞培养板,H9亚型禽流感病毒以不同剂量0(对照组)、10^-3(高剂量组)、10^-4(中剂量组)、10^-5×EID₅₀(低剂量组)分别接种,检测H9亚型禽流感病毒对MDCK细胞内抗氧化功能的影响。结果表明,与对照组相比,高剂量H9亚型禽流感病毒接种MDCK细胞使细胞内过氧化氢(H₂O₂)和羟基自由基(·OH)含量均显著增加(P>0.05),细胞内超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力均显著下降(P>0.05),细胞内的丙二醛(MDA)含量显著增加(P>0.05)。说明H9亚型禽流感病毒可显著提高MDCK细胞内活性氧自由基(ROS)含量,并降低抗氧化酶活力,阻碍ROS在细胞内的清除机制,引起细胞脂质过氧化作用,从而损伤细胞。     

Use of a staphylococcal vaccine to reduce prevalence of mastitis and lower somatic cell counts in a registered Saanen dairy goat herd

This investigation evaluated the efficacy of a bacterin in reducing the prevalence of staphylococcal mastitis and somatic cell counts (SCC) in a dairy goat herd. Does were vaccinated or left as controls, and the levels of mastitis and SCC monitored over 18 months. Staphylococcus caprae (42.5%), S. xylosus (15.1%), and S. simulans (10.0%) were the predominant causes of intramammary infections (IMI). The infection rate was 1.64 IMI/doe among vaccinates, which tended to be lower (P < 0.12) than controls (2.67 IMI/doe). The spontaneous cure rate of IMI after immunization was 1.28 cures/doe in vaccinates, which was higher than controls (0.6 cures/doe; P < 0.043). Average SCC of milk samples from vaccinates tended to be lower than that of controls (1274 × 10³/ml vs. 1529 × 10³/ml, respectively) (P < 0.10). Results support the continued study of mastitis vaccines for use in managing staphylococcal mastitis and SCC in dairy goats.     

禽流感病毒分子生物学的研究进展

禽流行性感冒(av ian in fluenza,A I,简称禽流感)是由A型禽流感病毒(av ian in fluenza v irus,A IV)引起的禽类烈性传染病。作为被世界动物卫生组织(O IE)定为A类的传染病,A I不仅给世界养禽业造成了巨大的经济损失,而且对人类健康和生命安全构成了严重威胁。因此,A I已经成为人们关注的焦点,国内外学者也对其进行了大量研究。作者从病原基因组及其编码的蛋白质、致病力、变异性以及对人类感染A IV的分子机制等角度就A IV的分子生物学研究作一综述,为防制A I提供理论基础,并在此基础上探讨了人类禽流感的防治措施,加深人们对A I的认识。