应用重组噬菌体抗体技术，从小鼠抗牛精子sp18蛋白的A5杂交瘤细胞系中分离总RNA，构建重组噬菌体抗体库。以牛精子作为包被抗原，经过三轮“亲和吸附-洗脱-扩增”淘选后，用phage-ELISA筛选出特异性阳性克隆。取其中特异结合牛精子细胞...应用重组噬菌体抗体技术，从小鼠抗牛精子sp18蛋白的A5杂交瘤细胞系中分离总RNA，构建重组噬菌体抗体库。以牛精子作为包被抗原，经过三轮“亲和吸附-洗脱-扩增”淘选后，用phage-ELISA筛选出特异性阳性克隆。取其中特异结合牛精子细胞的重组噬菌体抗体pCSA1克隆进行序列测定。将其ScFv(single chain fragment variable)因克隆到pET-30a(+)上，用IPTG进行诱导表达，成功得到了抗sp18单链抗体基因蛋白。展开更多
免疫印迹显示,抗牛精子sp18族膜蛋白的单克隆抗体（sp18单抗）与小鼠精子14,18,22,30和60 ku 5条带有较弱的抗原信号,同时,sp18单抗也能结合鸡卵溶菌酶。间接免疫荧光证实小鼠精子头后部存在sp18抗原。获能2h的小鼠精子与0.182 mg/mL的s...免疫印迹显示,抗牛精子sp18族膜蛋白的单克隆抗体（sp18单抗）与小鼠精子14,18,22,30和60 ku 5条带有较弱的抗原信号,同时,sp18单抗也能结合鸡卵溶菌酶。间接免疫荧光证实小鼠精子头后部存在sp18抗原。获能2h的小鼠精子与0.182 mg/mL的sp18单抗共孵育15～20min后进行体外受精,结果发现,单抗组的受精率（77.1％）与血清对照组（79.2％）和空白对照组（80.3％）间无显著差异（p>0.05）。继续培养受精卵发现,3组胚胎发育至2-细胞期的比率分别为100％,100％和97.9％（p>0.05）;发育至4-细胞期的比率分别为0,64.1％和64.3％,即单抗组胚胎的发育被完全阻断在2-细胞期（p<0.001）。0.5％链霉蛋白酶消化除去阻断胚胎的透明带后,间接荧光定位发现,2个胚胎细胞表面均存在sp18抗原。0.182mg/mL的sp18单抗与体内受精的胚胎共培养时,胚胎发育至2-细胞期（95.2％vs 92.9％）和4-细胞期（70.5 ％ vs 77.9％）的比例与对照组无显著差异（p>0.05）。结果表明,小鼠精子sp18族膜蛋白在受精时掺入卵母细胞质膜系统,成为胚胎细胞表面具有功能的质膜成分,与突破胚胎发育的2-细胞期阻断现象有关。展开更多
Western blot analysis revealed that one IgG<sub>1</sub> monoclonal antibody （mAb） to sp18 family membrane proteins （Mr. 14, 16 and 18 ku） of bovine sperm reacted faintly with protein bands of 14,18, 22...Western blot analysis revealed that one IgG<sub>1</sub> monoclonal antibody （mAb） to sp18 family membrane proteins （Mr. 14, 16 and 18 ku） of bovine sperm reacted faintly with protein bands of 14,18, 22, 30 and 60 ku （reducing） in samples of mouse sperm. The mAb also reacted to protein of egg lysozyme. Using a laser confocal microscope, indirect immunofluorescence （IIF） showed that the sp18 antigens were present in the posterior head of murine sperm. In murine in vitro fertilization （IVF） and embryo development trails, a total of 426 oocytes from C<sub>57</sub>BL/6 and F<sub>1</sub> hybrid strain (CD<sub>1</sub>×C<sub>57</sub>BL/6 cross) of 12, female mice were used in 3 independent trails. After preincubating capacitated sperm with 182 μg/mL of sp18 mAb in the modified TYH IVF medium for 15—20 min, cumulus-oocyte-complexes were introduced. The fertilization rate in sp18 mAb groups was 77.1%, which was not significantly （P】0.05） different from the nonspecific mouse IgG（79.2 %） and non-IgG（80.3%） control groups. Fertilized oocytes had been展开更多
To study the central mechanism of acupuncture stimulation of Sanyinjiao （三阴交 SP 6） in relieving dysmenorrhea. Methods： A total of 6 dysmenorrhea volunteer patients were subjected into this study. On the first po...To study the central mechanism of acupuncture stimulation of Sanyinjiao （三阴交 SP 6） in relieving dysmenorrhea. Methods： A total of 6 dysmenorrhea volunteer patients were subjected into this study. On the first positron emission tomography （PET） scan examination, they were assigned to pseudoacupuncture group by using the acupuncture needle just to prick the skin of Sanyinjiao （SP 6） ; while on the second PET scans, they were assigned to acupuncture group by inserting the needle into the same acupoint. 18F fluorodeoxyglucose （;SF-FDG） PET of the whole brain was performed during pseudo-acupuncture and real acupuncture of Sanyinjiao （SP 6）. The acquired PET data were analyzed by using statistical parametric mapping （SPM） software to determine changes of glucose metabolism in different cerebral regions. The patient＇s pain intensity was rated by using 0-10 numerical pain intensity scale. Results： After pseudo-acupuncture stimulation of Sanyinjiao （SP 6）, no significant changes were found in the pain intensity （ P 〉0.05）, while after real-acupuncture stimulation, the pain intensity declined significantly （ P〈0.01 ）. Following acupuncture of the right Sanyinjiao （SP 6）, multiple cerebral regions involving pain were activated （increase of glucose metabolism）, including ipsilateral lenticular nucleus （globus palliclus, putamen）, ipsilateral cerebellum and insular lobe, bilateral dorsal thalamus, ipsilateral paracentral Iobule, bilateral amygdaloid bodies, contralateral substantia nigra of the midbrain, bilateral second somatosensory （S Ⅱ ） areas, ispsilateral hippocampal gyms, frontal part of the ipsilateral cingulated gyrus, and bilateral mammary bodies of the hypothalamus. In addition, fewer regions of the cerebral cortex responded with decrease of the glucose metabolism after real acupuncture.展开更多
A las-like quorum-sensing system in Pseudomonas sp.M18 was identified,which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator.Several functions of the las system from strain M18 were investigated in t...A las-like quorum-sensing system in Pseudomonas sp.M18 was identified,which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator.Several functions of the las system from strain M18 were investigated in this study.The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin(Plt)and phenazine-1-carboxylic acid(PCA)by 4-5 fold and 2-3 fold over that of the wild type strain of M18,respectively.Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene.Expression of the translational fusions pltA'-'lacZ and phzA'-'lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons,and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.展开更多
The pltR gene,coding a putative LysR-type regulator,was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp.M18 using bioinformatics technology.The null mutation of pltR resulted in mutant M18TRG(pltR:...The pltR gene,coding a putative LysR-type regulator,was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp.M18 using bioinformatics technology.The null mutation of pltR resulted in mutant M18TRG(pltR::Gm)by recombination and its Plt(Pyoluteorin)production declined to 30%while PCA(Phenazine-1-carboxylic acid)production remained unchanged as compared with the wild-type M18 grown in King’s Medium B.After complementation,Plt production of mutant M18TRG was restored to the level in wild-type M18.Overexpression of pltR in M18 led to 13-fold enhancement of Plt produc-tion over the wild-type M18 strain.However,PCA production was unchanged under this condition.These data suggested that PltR was a positive regulator on Plt production.Plt itself,however,could not regulate expression of pltR.Expression of the plt-lacZ transcriptional fusion in mutant M18TRG de-clined obviously as compared with the wild-type M18,which further proved that PltR could regulate expression of Plt biosynthetic genes at the transcriptional level.In addition,the investigation on the pltR expression in gacA mutant M18G and rsmA mutant M18R disclosed that PltR was involved in the positive regulation of gacA on Plt production while being excluded from the negative control caused by rsmA.展开更多
文摘应用重组噬菌体抗体技术，从小鼠抗牛精子sp18蛋白的A5杂交瘤细胞系中分离总RNA，构建重组噬菌体抗体库。以牛精子作为包被抗原，经过三轮“亲和吸附-洗脱-扩增”淘选后，用phage-ELISA筛选出特异性阳性克隆。取其中特异结合牛精子细胞的重组噬菌体抗体pCSA1克隆进行序列测定。将其ScFv(single chain fragment variable)因克隆到pET-30a(+)上，用IPTG进行诱导表达，成功得到了抗sp18单链抗体基因蛋白。
文摘免疫印迹显示,抗牛精子sp18族膜蛋白的单克隆抗体（sp18单抗）与小鼠精子14,18,22,30和60 ku 5条带有较弱的抗原信号,同时,sp18单抗也能结合鸡卵溶菌酶。间接免疫荧光证实小鼠精子头后部存在sp18抗原。获能2h的小鼠精子与0.182 mg/mL的sp18单抗共孵育15～20min后进行体外受精,结果发现,单抗组的受精率（77.1％）与血清对照组（79.2％）和空白对照组（80.3％）间无显著差异（p>0.05）。继续培养受精卵发现,3组胚胎发育至2-细胞期的比率分别为100％,100％和97.9％（p>0.05）;发育至4-细胞期的比率分别为0,64.1％和64.3％,即单抗组胚胎的发育被完全阻断在2-细胞期（p<0.001）。0.5％链霉蛋白酶消化除去阻断胚胎的透明带后,间接荧光定位发现,2个胚胎细胞表面均存在sp18抗原。0.182mg/mL的sp18单抗与体内受精的胚胎共培养时,胚胎发育至2-细胞期（95.2％vs 92.9％）和4-细胞期（70.5 ％ vs 77.9％）的比例与对照组无显著差异（p>0.05）。结果表明,小鼠精子sp18族膜蛋白在受精时掺入卵母细胞质膜系统,成为胚胎细胞表面具有功能的质膜成分,与突破胚胎发育的2-细胞期阻断现象有关。
文摘Western blot analysis revealed that one IgG<sub>1</sub> monoclonal antibody （mAb） to sp18 family membrane proteins （Mr. 14, 16 and 18 ku） of bovine sperm reacted faintly with protein bands of 14,18, 22, 30 and 60 ku （reducing） in samples of mouse sperm. The mAb also reacted to protein of egg lysozyme. Using a laser confocal microscope, indirect immunofluorescence （IIF） showed that the sp18 antigens were present in the posterior head of murine sperm. In murine in vitro fertilization （IVF） and embryo development trails, a total of 426 oocytes from C<sub>57</sub>BL/6 and F<sub>1</sub> hybrid strain (CD<sub>1</sub>×C<sub>57</sub>BL/6 cross) of 12, female mice were used in 3 independent trails. After preincubating capacitated sperm with 182 μg/mL of sp18 mAb in the modified TYH IVF medium for 15—20 min, cumulus-oocyte-complexes were introduced. The fertilization rate in sp18 mAb groups was 77.1%, which was not significantly （P】0.05） different from the nonspecific mouse IgG（79.2 %） and non-IgG（80.3%） control groups. Fertilized oocytes had been
文摘To study the central mechanism of acupuncture stimulation of Sanyinjiao （三阴交 SP 6） in relieving dysmenorrhea. Methods： A total of 6 dysmenorrhea volunteer patients were subjected into this study. On the first positron emission tomography （PET） scan examination, they were assigned to pseudoacupuncture group by using the acupuncture needle just to prick the skin of Sanyinjiao （SP 6） ; while on the second PET scans, they were assigned to acupuncture group by inserting the needle into the same acupoint. 18F fluorodeoxyglucose （;SF-FDG） PET of the whole brain was performed during pseudo-acupuncture and real acupuncture of Sanyinjiao （SP 6）. The acquired PET data were analyzed by using statistical parametric mapping （SPM） software to determine changes of glucose metabolism in different cerebral regions. The patient＇s pain intensity was rated by using 0-10 numerical pain intensity scale. Results： After pseudo-acupuncture stimulation of Sanyinjiao （SP 6）, no significant changes were found in the pain intensity （ P 〉0.05）, while after real-acupuncture stimulation, the pain intensity declined significantly （ P〈0.01 ）. Following acupuncture of the right Sanyinjiao （SP 6）, multiple cerebral regions involving pain were activated （increase of glucose metabolism）, including ipsilateral lenticular nucleus （globus palliclus, putamen）, ipsilateral cerebellum and insular lobe, bilateral dorsal thalamus, ipsilateral paracentral Iobule, bilateral amygdaloid bodies, contralateral substantia nigra of the midbrain, bilateral second somatosensory （S Ⅱ ） areas, ispsilateral hippocampal gyms, frontal part of the ipsilateral cingulated gyrus, and bilateral mammary bodies of the hypothalamus. In addition, fewer regions of the cerebral cortex responded with decrease of the glucose metabolism after real acupuncture.
基金the Science Program of the China National 10th"Five-year Plan"(Grant No.2004BAAA308A21-6)the National Natural Science Foundation of China(Grant No.3037004)+1 种基金the National High Technology Research and Development Program(863 Program)of China(Grant No.2007AA02Z215)New Century Excellent Talent Program(NCET)in China and Shanghai Leading Academic Discipline(Project Number:B203)
文摘A las-like quorum-sensing system in Pseudomonas sp.M18 was identified,which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator.Several functions of the las system from strain M18 were investigated in this study.The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin(Plt)and phenazine-1-carboxylic acid(PCA)by 4-5 fold and 2-3 fold over that of the wild type strain of M18,respectively.Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene.Expression of the translational fusions pltA'-'lacZ and phzA'-'lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons,and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.
基金Supported by the Science Program of the China National 10th"Five-year Plan"(Grant No.2004BA308A21-6)the National Natural Science Foundation of China(Grant No.30370041)and NCET in China
文摘The pltR gene,coding a putative LysR-type regulator,was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp.M18 using bioinformatics technology.The null mutation of pltR resulted in mutant M18TRG(pltR::Gm)by recombination and its Plt(Pyoluteorin)production declined to 30%while PCA(Phenazine-1-carboxylic acid)production remained unchanged as compared with the wild-type M18 grown in King’s Medium B.After complementation,Plt production of mutant M18TRG was restored to the level in wild-type M18.Overexpression of pltR in M18 led to 13-fold enhancement of Plt produc-tion over the wild-type M18 strain.However,PCA production was unchanged under this condition.These data suggested that PltR was a positive regulator on Plt production.Plt itself,however,could not regulate expression of pltR.Expression of the plt-lacZ transcriptional fusion in mutant M18TRG de-clined obviously as compared with the wild-type M18,which further proved that PltR could regulate expression of Plt biosynthetic genes at the transcriptional level.In addition,the investigation on the pltR expression in gacA mutant M18G and rsmA mutant M18R disclosed that PltR was involved in the positive regulation of gacA on Plt production while being excluded from the negative control caused by rsmA.
