微小牛蜱Bm91基因的原核表达

根据GenBank收录的微小牛蜱Bm91基因序列设计一对表达引物,以微小牛蜱幼蜱总RNA反转录合成的cDNA为模板,扩增获得了Bm91基因,其长度为1 908 bp,包含一个大小为1 833 bp的完整开放阅读框,编码611个氨基酸,该蛋白预期分子质量为83 ku。将Bm91基因片段亚克隆到原核表达载体pET-30a,构建重组原核表达载体pET-30a-Bm91,转化BL21宿主菌,经IPTG诱导,发现在0.8 mmol/LIPTG,诱导时间2 h,温度37℃条件下,目的重组蛋白表达量最大,约占蛋白总量的20%。SDS-PAGE检测表明,表达产物为分子质量为83 ku的融合蛋白,与预期大小一致;Western blot分析表明,表达产物能被兔抗微小牛蜱阳性血清所识别。     

贵州微小牛蜱的检测与防制研究

微小牛蜱在贵州某些奶牛场、人工草场、养牛专业户及农户的牛群中存在,严重危害牛群的健康.对贵州微小牛蜱的危害、流行情况、监测及防制进行了探索,可为进一步开展微小牛蜱的防治研究提供参考依据.     

The Effects of Salivary Gland Extracts from <Emphasis Type="Italic">Boophilus microplus</Emphasis> Ticks on Mitogen-stimulated Bovine Lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.     

溴氰菊酯对微小牛蜱的毒力测定和杀灭试验

溴氰菊酯对微小牛蜱未吸血幼蜱的半致死浓度（ＬＣ５０）为０．３６×１０－６，对未吸血成蜱的半致死量（ＬＤ５０）为４．６８７μｇ／虫，对饱血雌蜱的ＬＤ５０为６．８６８μｇ／虫；而敌百虫对未吸血幼蜱的ＬＣ５０为１７５×１０－４，对未吸血成蜱和饱血雌蜱的ＬＤ５０分别为１１１６和５２６６μｇ／虫．可见，溴氰菊酯对微小牛蜱未吸血幼蜱、未吸血成蜱及饱血雌蜱的杀蜱效果分别为敌百虫的４８６、２３８和７６７倍．现场杀蜱试验，采用５％溴氰菊酯水和剂１２．５×１０－４时．杀蜱率达９２％，３０×１０－６时达１００％杀蜱效果，对牛无副作用。     

福建省微小牛蜱的生物学特性

福州地区的微小牛蜱Boophilus microplus在牛体表终年均有寄生,以7、8、9、3个月为寄生高峰期。经人工饲养观察,每年发生4代。第一代需要63—88天,第二代42—56天,第三代44—62天,第四代90—152天。以第四代未吸血幼蜱或第三代未吸血幼蜱越冬。未吸血幼蜱最长能存活227天。雌蜱一生共产卵1589～4138粒(平均2986.3粒),产完卵后约0～14天死亡。饱血雄蜱一般存活不超过1个月。饱血雌蜱的体重与产卵量和产卵期呈相关[Y(产卵量)=00.945,P<0.01;r(产卵期)=0.490,p<0.05]。     

牛血孢子虫单一种的分离技术及其保藏方法的研究——卵形巴贝斯虫的传播试验

将实验室培育的“洁净”长角血蜱的幼虫、若虫、成虫及微小牛蜱的幼虫,先后分别释放到人工感染卵形巴贝斯虫单一种牛体上不同部位事先粘贴好的布袋中,使其自行叮咬吸血。待饱血脱落后亦分别收集,置28℃、相对湿度约90％的条件下蜕皮或产卵孵化。而后用不同世代和各变态期的蜱,分别叮咬感染除脾和非除脾健康易感牛。结果表明,长角血蜱的当代若虫和成虫对卵形巴贝斯虫没有传播能力,幼虫和若虫吸入的病原亦不能经卵传递;饱血雌虫吸入的病原可经卵传递,次代幼虫、若虫和成虫三个阶段都具有传播能力。次代感染幼虫经兔体后的若虫和成虫也具有感染力。 微小牛蜱不能传播该种病原。     

Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.     

Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.     

Characterization and Inhibitor Studies of Chitinases from a Parasitic Blowfly (Lucilia cuprina), a Tick (Boophilus microplus), an Intestinal Nematode (Haemonchus contortus) and a Bean (Phaseolus vulgaris)

The molecular weight pattern and the stage-specific activities of chitinases from the blowfly Lucilia cuprina, the tick Boophilus microplus and the intestinal nematode Haemonchus contortus were examined. Chitinolytic enzymes could be detected in all parasite species tested, but the activity was different between the stages. Highest chitinolytic titers were found in blowfly pupae (83 kDa, 118 kDa), hatching larvae of ticks (58 kDa, 94 kDa) and nematode eggs (43 kDa). Leaves from ethylene-treated bean plants Phaseolus vulgaris expressed two basic Class I chitinases (Ia, Ib) of 34 kDa, differing in their amino acid sequences at residue 33 and 34 (Ia: glycine, proline; Ib: lysine, aspartic acid). Inhibitor studies with blowfly pupae revealed that allosamidin (IC₅₀=0·32 (±0·02) μM ) was by far the best inhibitor when compared with various amino sugar derivatives. This compound also inhibited chitinases from tick larvae (IC₅₀=0·69(±0·10) μM ) and nematode eggs (IC₅₀=0·048(±0·0045) μM ) specifically. Whereas Class Ia chitinase from bean leaves was inhibited only up to 18% by 10 μM allosamidin, it had an IC₅₀ of 1(±0·14) μM for the Ib type, which is the first plant chitinase described to be highly sensitive to allosamidin.