B组轮状病毒WH-2株vp7基因的原核表达与抗体的制备

目的：在大肠杆菌中表达人B组轮状病毒WH-2株VP7蛋白并制备其兔抗血清。方法：根据B组轮状病毒（GBRV）WH-2株vp7基因的全序列设计引物,用PCR的方法扩增得到vp7基因的编码区。将其克隆到原核GST融合表达载体pGEX-KG内,转化大肠杆菌E.coliDH5α,IPTG诱导表达人B组轮状病毒WH-2株VP7蛋白,经SDS-PAGE分离纯化表达的蛋白免疫新西兰兔,制备人B组轮状病毒WH-2株VP7蛋白抗血清。结果：经鉴定确认,vp7基因以正确的方式插入到载体中,此重组表达载体经IPTG诱导后,可表达相对分子量为53.4 kDa的GST-VP7融合蛋白。制备的抗血清经同样诱导表达的表达载体pGEX-KG表达产物吸收后1：500倍稀释后用Western Blot分析,与53.4 kDa的GST-VP7融合蛋白获得特异性显色信号。结论：人B组轮状病毒WH-2株VP7蛋白成功在大肠杆菌中GST融合表达,所表达的蛋白和制备的抗体不但为研究结构与功能提供了物质基础,也为GBRV所引起的疾病预防、诊断和治疗等流行病学研究和临床诊断奠定了基础,具有重要实际应用价值。     

Analgesic actions of dynorphin A(1–13) antiserum in the rat brain stem

This study was performed to evaluate the effects of dynorphin A(1–13) antiserum when microinjected into an active hyperalgesic region within the rat brain stem. When administered within the dorsal posterior mesencephalic tegmentum (DPMT) of intact conscious rats, dynorphin A(1–13) antiserum produced rapid onset and persistent prolongation of a low intensity thermally evoked tail avoidance response (LITETAR). These analgesic actions of the dynorphin A(1–13) antiserum appeared to be dose dependent. These studies support previous hypotheses about the existence of tonically active brain stem opioid hyperalgesic processes. Further, the results provide indirect evidence for a potential role of brain stem dynorphin(s) in facilitating pain.     

延髓—脊髓P物质能神经元下行通路参与毒扁豆碱的中枢升压反应

大鼠延髓腹侧面头端应用毒扁豆碱引起血压升高和心率加快,伴有延髓腹侧面头端胆碱酯酶活性降低和脊髓蛛网膜下腔灌流液中P物质样免疫反应活性升高。在延髓腹侧面头端应用阿托品或脊髓蛛网膜下腔注射P物质拮抗剂D-脯~2,D-苯丙~7,D-色~9-P物质均可阻断毒扁豆碱的心血管效应。脊髓蛛网膜下腔注射P物质抗血清或辣椒素均可减弱毒扁豆碱的升压反应。实验结果提示,毒扁豆碱作用于延髓腹侧面头端的M受体,兴奋了延髓-脊髓P物质能神经元下行通路,使之释放P物质,引起交感肾上腺髓质系统兴奋,从而使血压升高和心率加快。     

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

兔BK通道β亚基多克隆抗血清的制备

目的:用小鼠制备抗兔BK通道β亚基的抗血清。方法:采用RT-PCR扩增编码兔BK通道β亚基胞内段的基因。在大肠杆菌中表达GST-β亚基融合蛋白。以从PAGE凝胶上切下的融合蛋白免疫BALB/c小鼠制备多克隆抗血清。用ELISA和Westernblot鉴定抗血清的特异性。结果:用RT-PCR扩增出约300bp的兔BK通道基因。序列分析显示,扩增的序列与已发布的新西兰大白兔骨骼肌BK通道的序列完全一致。在E.coliDH5α成功地表达Mr约为37000GST-β亚基融合蛋白。ELISA和Westernblot检测证明,针对GST-β亚基融合蛋白的抗血清,不仅可特异性地识别GST-β,也可识别源于兔组织的β蛋白。抗血清的最高滴度达1∶128000。结论:克隆了编码兔BK通道β亚基胞内段的基因,并成功地获得可特异性识别新西兰大白兔BK通道β亚基的高滴度抗血清,为BK通道的进一步研究提供了物质基础。     

受体－A蛋白融合蛋白制备抗P物质受体血清及其特性

受体－Ａ蛋白融合蛋白制备抗Ｐ物质受体血清及其特性ＷａｌｔｅｒＫ．Ｋ．Ｈｏ（何国强）；ＡｌｅｘｉｓＰ．Ｋ．Ｃｈｅ，郑德枢（香港中文大学；香港生物化学系，香港沙田；广州医学院神经科学研究所，广州５１０１８２）本文报告了用直肠杆菌表达Ｐ物质受体－Ａ蛋白融合...     

小鼠ZP2抗体的制备及其免疫活性研究

目的制备抗小鼠ZP2（mZP2）抗体并研究抗血清的免疫活性。方法利用Rosetta宿主菌诱导表达mZP2重组蛋白,电泳
分离重组ZP2蛋白,切胶制备免疫抗原;利用乳化抗原主动免疫新西兰雄兔制抗血清;通过ELISA测定抗体应答水平;以免疫组
化测定抗血清特异性;通过精卵结合实验检测抗血清对小鼠卵子结合顶体反应后精子的影响。结果ELISA结果说明用重组蛋
白免疫新西兰雄兔诱发产生了抗血清。免疫组化实验证明抗血清与小鼠卵巢透明带发生了特异性反应,精卵结合实验显示抗
ZP2抗体能抑制顶体反应后精子与卵子结合。结论重组mZP2蛋白诱发产生了抗ZP2抗体,抗体能抑制顶体反应后的精子与
卵子结合。
     

Effect of prolactin and prostaglandins on the stimulation of prolactin binding sites in the male rat liver

Induction of prolactin hepatic receptors in the male rat by exogenous prolactin and the possible involvement of prostaglandins in this process were studied. When ovine prolactin 500 microgram/kg was administered sc in saline containing 10% PVP (polyvinylpyrrolidone), twice daily for 6.5 days and the rats killed 48 h after the last injection, the specific binding of 125I-labelled ovine prolactin to prolactin hepatic receptors was raised 26-fold, while administration of prolactin in saline only caused a 7-fold increase. A well correlated log dose-response relationship was demonstrated between 12.5 and 500 microgram of prolactin in saline-PVP, with lowest dose causing an 18-fold increase in binding. A shorter 4.5 day treatment of prolactin in saline-PVP caused only a small 3-fold increase in prolactin binding. Scatchard analysis showed that these increases resulted from increases in receptor concentration. The effect of prolactin on the induction of the hepatic receptors could not be mimicked by PGE1, PGE2 or PGF2 alpha, nor could PGF2 alpha synergize with a short treatment with prolactin. Further, indomethacin caused no significant effect on this action of prolactin. It seems that prolactin does not induce its own receptors in the rat liver by stimulation of prostaglandins in this tissue.     

Endoxin Antagonist Lessens Myocardial Ischemia Reperfusion Injury

OBJECTIVE: To elucidate whether endoxin is one of important factors involved in myocardial ischemia reperfusion (MIR) injury, the change of myocardial endoxin level was determined in rats with MIR injury model and the effects of anti-digoxin antiserum (ADA), an endoxin specific antagonist, on MIR injury were studied. METHODS: MIR injury model was obtained by ligating left anterior descending coronary artery 30 min followed by 45 min reperfusion. Sprague-Dawley rats were randomly divided into six groups of 10 rats, each. Sham group, MIR group, normal saline group, ADA 9, 18 and 36 mg.kg(-1). ECG was continuously recorded. After reperfusion left ventricular myocardium samples of ischemic area were processed immediately. Myocardial endoxin level, Na(+)-K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase activities, and intramitochondrial Ca(2+) content were measured. RESULTS: Myocardial endoxin level was significantly increased; Na(+)-K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase activities were remarkably decreased; intramitochondrial Ca(2+) content was remarkably raised; ST segments of ECG were significantly elevated and occurrence and scores of ventricular arrhythmias were significantly increased in early stage of reperfusion in rats with MIR. In all groups with ADA, myocardial endoxin level was remarkably decreased; Na(+)-K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were drastically increased; intramitochondrial Ca(2+) content was declined; ST segments and ventricular arrhythmias were improved. CONCLUSION: Myocardial endoxin level was increased in MIR, which implies that the elevated endoxin may be one of major factors inducing MIR injury. This postulate is supported by the observation that ADA has protective and therapeutic effects against MIR injury probably by antagonizing the action of endoxin. The underlying mechanism may be ascribed to restoration of energy metabolism, and attenuation of intracellular Ca(2+) overload.     

兔抗小鼠胚胎干细胞免疫血清对8-细胞胚胎发育的影响

目的 制备兔抗小鼠胚胎干细胞免疫血清,观察其对小鼠胚胎发育的影响。方法 用小鼠胚胎培养基稀释兔抗血清,分别稀释至50倍、100倍、200倍,用其对小鼠8-细胞胚胎进行培养,通过胚胎致密化、囊胚率、胚胎体外贴壁生长、细胞计数、胚胎移植和免疫荧光染色观察胚胎发育状况。 结果 形态学上50倍稀释的抗血清能够抑制8 细胞胚胎的发育甚至使其死亡;100倍稀释的抗血清能够延迟8 细胞胚胎的致密化,并使得胚胎在囊胚化过程中出现空泡化的现象;200倍稀释的抗血清作用不明显。未作免疫处理的兔血清与空白对照组差异不显著。100倍稀释的抗血清能够显著抑制囊胚内细胞团（ICM）的发育、囊胚细胞分布紊乱、胚胎细胞数目减少以及囊胚畸形率增加。虽然囊胚的碱性磷酸酶和Oct4均呈阳性,但经胚胎移植后都不能正常发育。 结论 兔抗小鼠胚胎干细胞免疫血清能够抑制小鼠胚胎致密化过程和囊胚内细胞团的发育,使囊胚出现空泡化,细胞分布紊乱,抑制胚胎着床。