产气荚膜梭菌α毒素基因工程菌表达产物的免疫原性研究

已构建的能表达产气荚膜梭菌α毒素保护性抗原基因工程菌株Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）经动物试验证实没有毒性。从ＩＰＴＧ诱导后的工程菌中提取包涵体,再辅以氢氧化铝胶制成抗原,免疫小鼠３０ｄ后,用产气荚膜梭菌强毒株培养上清及培养菌体攻击,结果免疫鼠能抵抗至少２ＬＤ１００的攻击,证明Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）工程菌株表达产物具有良好的免疫原性。     

产气荚膜梭菌α毒素基因工程菌表达产物免疫原性研究

已构建的能表达产气荚膜梭菌α毒素保护性抗原基因工程菌株Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）经动物试验证实没有毒性。从ＩＰＴＧ诱导后的工程菌中提取包涵体,再辅以氢氧化铝胶制成抗原,免疫小鼠３０ｄ后,用产气荚膜梭菌强毒株培养物上清及培养菌体攻击,结果免疫鼠能抵抗至少２ＬＤ１００的攻击,证明Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）工程菌株表达产物具有良好的免疫原性。     

表达大肠杆菌肠毒素ST1-LTB融合蛋白双价基因工程菌株的构建

重组菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）经ＩＰＴＧ诱导后,其表达产物经ＳＤＳ－ＰＡＧＥ和ＥＬＩＳＡ检测,结果表明重组菌株可以高效表达大肠杆菌肠毒素ＳＴ１－ＬＴＢ 融合蛋白,该融合蛋白占菌体总蛋白的３３．２１％ ,而且已失去天然ＳＴ１肠毒素生物毒性。用从ＩＰＴＧ 诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原免疫小鼠,结果免疫小鼠至少能抵抗１．５ＭＬＤ 的大肠杆菌强毒株Ｃ８３９０２（Ｋ８８ａｃ,ＳＴ＋ ,ＬＴ＋ ）的攻击。用提取的包涵体免疫家兔后,采集的血清能够中和天然ＳＴ１肠毒素的毒性。这表明构建的工程菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株     

大肠杆菌肠毒素ST1-LTB融合基因的高效表达

用限制性核酸内切酶ＥｃｏＲⅠ和ＨｉｎｄⅢ双酶切含大肠杆菌肠毒素ＳＴ１—ＬＴＢ融合基因的质粒ｐＸＳＬＴ１,回收０８４ｋｂ的ＳＴ１—ＬＴＢ融合基因,再将载体ｐＥＴ—２８ｂ（＋）用ＥｃｏＲⅠ和ＨｉｎｄⅢ双酶切,然后将其与ＳＴ１—ＬＴＢ融合基因连接,转化至受体菌ＢＬ２１（ＤＥ３）中。经ＥｃｏＲⅠ、ＨｉｎｄⅢ、ＢａｍＨⅠ酶切反应鉴定重组子质粒,得到了理想重组质粒ｐＸＥＴＳＬＴ１。重组菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）经ＩＰＴＧ诱导后,其表达产物经ＳＤＳ—ＰＡＧＥ和ＥＬＩＳＡ检测,结果表明重组菌株可以高效表达ＳＴ１—ＬＴＢ融合蛋白,该融合蛋白占菌体总蛋白的３３２１％,而且无天然ＳＴ１生物毒性。     

狂犬病病毒糖蛋白基因在原核细胞中的表达

将狂犬病病毒糖蛋白（ＲＶｇｐ）基因ＢｇｌⅡ片段（１６７５ｂｐ）分别正向插入到原核高效表达载体ｐＥＴ－１７ｂ和ｐＥＴ－１７ｂ２（用ＳａｃⅠ－ＮｄｅⅠ缺失掉ｐＥＴ－１７ｂ６０ｂｐ含起始密码子ＡＴＧ小片段）的ＢａｍＨⅠ切点，构建重组质粒ｐＥＴ－１７ｂＲＶｇｐ和ｐＥＴ－１７ｂ２ＲＶｇｐ。将其分别转化表达受体菌Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）和Ｅ，ｃｏｌｉＢＬ２１（ＤＥ３）ｐｌｙｓＳ．ＩＰＴＧ诱导表达，菌体经超声波裂解处理后ＳＤＳ－ｐＡＧＥ，染色，在分子量约６００００处可见重组质粒表达的较宽的蛋白带，以抗ＲＶｇｐＭｃＡｂ进行Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测，表明该表达蛋白为ＲＶｇｐ。通过扫描显示，表达的ＲＶｇｐ占菌体总蛋白的１０％～１４％，其中ｐＥＴ－１７ｂ２ＲＶｇｐ在Ｅ。ｃｏｌｉＢＬ２１（ＤＥ３）中的表达量最高。     

大肠杆菌肠毒素ST1—LTB融合蛋白工程菌株的免疫原性研究

已构建的能表达大肠杆菌肠毒素ＳＴ１ＬＴＢ融合蛋白的工程菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）及其表达产物经动物试验证实没有毒性反应。用从ＩＰＴＧ诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原,免疫小鼠,结果免疫小鼠至少能抵抗１５ＭＬＤ的大肠杆菌强毒株Ｃ８３９０２（Ｋ８８ａｃ,ＳＴ＋,ＬＴ＋）的攻击。用提取的包涵体免疫家兔后,采集的血清能够中和天然ＳＴ１的毒性。这表明构建的工程菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株。     

绿脓杆菌外毒素A受体结合亚单位基因的克隆与表达

为防治绿脓杆菌感染造成的细胞坏死及多器官衰竭，本试验克隆了含绿脓杆菌外毒素Ａ（ＰＥＡ）结合亚单位（Ⅰ区）和部分跨膜亚单位（Ⅱ区）编码３０９个氨基酸的约１０００ｂｐ的基因片段，以正确阅读框架将该片段置于原核高效表达Ｔ７启动子下游，构建了高效表达质粒ｐＥＴ－ＥＡＢ。转化宿主菌ＢＬ２１（ＤＥ３）、ＨＭＳ１７４（ＤＥ３）、ＨＭ１７４（ＤＥ３）ｐｌｙｓＳ和ＢＬ２１（ＤＥ３）ｐｌｙｓＳ后，经ＩＰＴＧ诱导４ｈ，均表达了外源目的基因蛋白，其中ＢＬ２１（ＤＥ３）和ＢＬ２１（ＤＥ３）ｐｌｙｓＳ的表达量明显高于其他宿主菌。经ＳＤＳ－ＰＡＧＥ分析，外源蛋白分子量约３４０００，与ＰＥＡⅠ区和部分Ⅱ区基因所编码的蛋白理论估计值相符，命名为ＰＥ３４。凝胶薄层扫描显示，ＰＥ３４含量占菌体蛋白总量的５６％。Ｗｅｓｔｅｒｎ－ｂｌｏｔｉｎｇ检测证明ＰＥ３４为所需目的蛋白     

类志贺氏菌毒素Ⅱ型变异体B亚单位基因的表达与鉴定

将由 Ｐ Ｃ Ｒ 扩增并克隆在ｐ Ｕ Ｃ１８ 质粒载体中的ｓｌｔⅡｅ Ｂ基因切出,并按预定的阅读框架插入表达性质粒载体 ｐ Ｇ Ｅ Ｘ６ｐ１ 中的谷胱苷肽转移酶（ Ｇ Ｓ Ｔ）基因的下游。重组质粒 ｐｐｐｓｌｔⅡｅ Ｂ在大肠杆菌 Ｂ Ｌ２１ 中以融合蛋白的形式表达 Ｓ Ｌ ＴⅡｅ Ｂ蛋白（命名为 Ｇ Ｓ Ｔ Ｓ Ｌ ＴⅡｅ Ｂ）,即 Ｓ Ｌ ＴⅡｅ Ｂ蛋白（７５６８ｋ Ｄ）与谷胱苷肽转移酶（２７３３５ｋ Ｄ）相连组成分子量为 ３４８８５ｋ Ｄ 的融合蛋白。通过对重组大肠杆菌 Ｐ Ｐ Ｓ Ｌ ＴⅡｅ Ｂ的菌体裂解物的 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳分析,以及根据抗原抗体结合反应的免疫学特性,通过 Ｗｅｓｔｅｒｎｂｌｏｔ 鉴定,重组大肠杆菌 Ｐ Ｐ Ｓ Ｌ ＴⅡｅ Ｂ（含有重组质粒 ｐｐｓｌｔⅡｅ Ｂ的大肠杆菌 Ｂ Ｌ２１）可以大量表达融合蛋白形式的 Ｓ Ｌ ＴⅡｅ Ｂ。根据 Ｇ Ｓ Ｔ 可与其底物谷胱苷肽结合的特性,用谷胱苷肽结合的 Ｓｅｐｈａｒｏｓｅ ４ Ｂ制备的亲和凝胶纯化表达产物,纯化的表达产物经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ,可以鉴定到分子量约为 ３５ｋ Ｄ 的蛋白条带,这与融合蛋白形式的 Ｓ Ｌ ＴⅡｅ Ｂ分子量（３４８８５ｋ Ｄ）相当。     

产气荚膜梭菌α毒素保护性抗原基因的克隆与核苷酸序列分析

将含产气荚膜梭菌α毒素基因的质粒ｐＫＭＡ１００用限制性核酸内切酶ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,经１．５％琼脂糖凝胶电泳分离、透析袋电洗脱法回收０．９５ｋｂα毒素基因片段,再将载体ｐＥＴ－２８ｂ（＋）用ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,然后将处理好的ｐＥＴ－２８ｂ（＋）与０．９５ｋｂ的α毒素基因片段通过Ｔ４ＤＮＡ连接酶进行粘性末端连接,转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨＩ和ＨｉｎｄⅢ双酶切分析和核苷酸序列分析,证明重组质粒ｐＸＥＴＡ１含有产气荚膜梭菌α毒素基因。确定了α毒素基因的全部核苷酸序列,并证明其具有正确的阅读框架。     

表达CPB—ST融合蛋白工程菌株的免疫原性研究

已构建的能表达产气荚膜菌β－毒素和大肠埃希氏菌ＳＴ融合蛋白的基因工程菌株ＢＬ２１（ＤＥ３,ｐＥＣＢ－ＳＴ１）及其表达产物经动物试验证实没有毒性反应。用从ＩＰＴＧ诱导的工程菌株中提取的包涵体和经甲醛灭活的工艺菌株全菌体制备抗原免疫小白鼠,攻毒试验结果表明,免疫小白鼠能分别抵抗１ＭＬＤ的Ｂ型产气荚膜梭菌强毒株和产ＳＴ的大肠埃希氏菌工程菌株ＨＢ１０１（ｐＳＬＭ００４）的攻击。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.