目的 建立一种简便、实用、准确的检测5,10-亚甲基四氢叶酸还原酶(MTHFR)基因C677T位点多态性的方法 .方法在分析MTHFR基因C677T多态位点相关序列的基础上,设计1对特异性引物,通过聚合酶链反应扩增1个535 bp的靶片段,该靶片段跨越MTHFR基因C677T多态位点,且在多态位点上游163 bp处含有一个限制性核酸内切酶HinfI识别位点,该识别位点可作为后续HinfI酶切分型的内对照,PCR产物用HinfI快速消化,再用琼脂糖凝胶电泳分离消化产物,得出相应的限制性酶切图谱用于判别基因型.结果 可从限制性酶切图谱中明确判断各样品MTHFR基因C677T位点基因型,CC、CT、TT 3种基因型样品的分型结果与基因测序完全吻合.采用本法检测了100例样品,检得MTHFR C677T 3种基因型样品即CC、CT、TT的频率分别为0.59、0.35、0.06;C和T等位基因频率分别为0.765和0.235,符合Hardy-Weinberg遗传平衡(χ2=0.0723,P=0.965>0.05).结论 该方法由于PCR产物中含有HinfI酶切内对照序列,能克服传统PCR-RFLP法酶切不完全可能导致基因型误判的缺点,简便、实用、准确,适合一般实验室应用及流行病学调查.

Activity and half-life play key roles in the application of GHRH analogues. The GHRH monomers produced in a solid synthesizer were incubated, respectively, in NH4OH solution and lyophilized to obtain their dimers. The activities, specificities, and receptor affinities of the GHRH dimers were evaluated in rGH release/inhibition, rACTH/LH/PRL release, pituitary homogenate binding, and fluorescent staining. Compared to hGHRH(1-44)NH2 (S), PP-hGHRH(1-44)-GGC-CGG-hGHRH(44-1)-PP (2D), P-hGHRH(1-44)-GGC-CGG-hGHRH(44-1)-P (2E), 1P-hGHRH(2-44)-GGC-CGG-hGHRH(44-2)-1P (2F), or hGHRH(1-44)-GGC-CGG-hGHRH(44-1) (2Y) had potency of 104 ± 16.7%, 94 ± 32.6%, 114 ± 16.6%, or 122 ± 14.5% and similar specificities. The inhibition effect of GHIH on rGH stimulated by GHRH dimer was in dose-/time-dependent manner. The staining of FITC-labeled dimer showed cytomembrane distribution and the binding ranking was 2F>2D>2Y>2E>S. 2F presents the strongest activity and the highest affinity to pituitary cells. The dimer with 1Pro-GHRH stimulates stronger rGH release than that with 1Tyr-GHRH and the N-terminal single cyclic amino acid is required for the stimulation.

目的 研究环磷酰胺对雄鼠生殖系统的影响,为雄性不孕模型的建立提供实验参考.方法 以雄性昆明小鼠、SD大鼠和中华仓鼠为研究对象,每种动物分为生理盐水组和环磷酰胺组.小鼠腹腔给药200 mg/(kg·周),共5周;大鼠灌胃给药40 mg/(kg·d),共4周;仓鼠腹腔给药30 mg/(kg·周),共5周.各动物每周称质量1次,观察活动情况,计算存活率.建模后,观察睾丸镜下形态学变化.结果 环磷酰胺组动物活动度变差,毛发变稀疏,体质量明显降低.小鼠、大鼠和仓鼠环磷酰胺组存活率分别为100％、20％(P＜0.01)和80％ (P＜0.05).镜下检查发现环磷酰胺组睾丸曲精管有空泡,生精细胞排列混乱,管腔内精子减少.结论 环磷酰胺可明显引起实验动物睾丸组织损伤.昆明小鼠对环磷酰胺耐受性最高,但睾丸太小,不易观察.SD大鼠非常敏感,死亡率很高.中华仓鼠耐受性良好,睾丸体积较大,适合建立不育模型.

数学思想是数学活动的指导思想,数学活动的一般概括.它从整体和思维的更高层次上指导学生有效地认识数学的本质,运用数学知识发现、完善数学知识结构,探寻解题的方向和途径.函数是高中数学的主线,它用联系和运动、变化的观点研究、描述客观世界中相互关联的量之间的依存关系.函数思想以函数知识做基石,用运动变化的观点分析和研究数学对象间的数量关系,丰富并优化数学解题活动.

目的 通过生长激素释放激素肽(GHRH)改构寻找新的人生长激素释放激素类似肽(hGHRH Analog).方法 以固相合成技术合成6种新型N端和(或)C端改构的GHRH类似肽Pro-Pro-hGHRH(1-44)、Pro-hGHRH(1-44)、1pro-GHRH(2-44)、Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys、Pro-hGHRH(1-44)-Gly-Gly-Cys和1Pro-GHRH (2-44)-Gly-Gly-Cys.用C18反相高效液相色谱法和电喷雾质谱法检验其纯度和精确分子量.通过大鼠垂体刺激实验测定这些肽的鼠生长激素和其他垂体激素的释放值.结果 通过N端Pro-Pro-或Pro-延伸,1Pro-代换,和(或)C端Gly-Gly-Cys延伸获得的新型人GHRH类似肽活性有明显结构-功能关系:Pro-Pro-延伸＜Pro-延伸＜1Pro-代换;与标准的hGHRH(1-40)比较,1Pro-hGHRH(2-44)-Gly-Gly-Cys肽达到了相似的GH释放活性;其他垂体激素释放活性显示,6种新型类似肽均具有一定的垂体激素释放特异性.结论 这种GHRH肽结构改构获得具有一定生物学活性的新类似肽,为未来深入研究结构-活性关系,打下良好的基础.

Entomopathogenic Photorhabdus bacteria produce two types of intracellular crystalline inclusion proteins,CipA and CipB,as the nutrient resource for the symbiotic nematode.Based on constructed high-level prokaryotic expression system,recombinant Cip proteins were purified with a warm extract method and used as the antigens to immune New Zealand rabbits,respectively.The polyclonal antibodies with the titers of 1∶6 400 were raised and monitored by indirect enzyme-linked immunosorbent assay(ELISA).Western blot assay confirmed that the antibodies could specifically recognize the inclusion proteins in Photorhabdus bacteria.Preparation of Cip polyclonal antibodies will be helpful to develop the immunological assay method of these proteins.

Objective: To observe the expression and processing of carboxypeptidase E (CPE) mediated neuroendocrinopeptide in human breast adenocarcinoma. Methods: By using RT-PCR, immunohistochemistry, ELISA, and Western blot techniques, the expression profiles of CPE and growth hormone releasing hormone (GHRH) gene and protein were investigated between 42 human breast tumor tissues and 21 tumor-adjacent normal ones. Results: The expression level of CPE gene in the breast adenocarcinoma tissues was 1024±237, while it was 805±83 in the tumor-adjacent normal tissues (P<0.01). The CPE protein in the breast tumor tissues showed higher expression than in the tumor-adjacent normal tissues (P<0.01). The expression level of GHRH gene in the breast adenocarcinoma tissues was 1522±457, while it was 1067±437 in the tumor-adjacent normal tissues (P<0.01). The GHRH protein was up-regulated in the tumor tissues (P<0.01). Conclusions: The high expressions of CPE and GHRH suggest that there is a strong CPE mediated neuroendocrinopeptide posttranslational processing in the breast tumor cells.

Background: The processing of proprotein convertase (PC)-mediated neuropeptide plays a very important role in carcinogenesis and tumor proliferation. Aim: To investigate proneuropeptide processing mechanism in tumorigenesis and tumor proliferation. Materials and methods: The expression and processing profiles of PC1, carboxypeptidase E (CPE), PC2, GHRH, or neuropeptide Y (NPY) gene and protein level were investigated between 42 human breast tumor tissues and 21 tumor-adjacent normal tissues. Results: Gene analyses indicated that the proPC1, CPE , or preproNPY gene had higher expression in the breast tumor tissues, whereas the proPC2 or preproGHRH gene showed lower expression in the tissues. Protein analyses showed that the proPC1, PC1, CPE, GHRH, and preproNPY proteins were up-regulated in the tumor tissues, whereas the proPC2, PC2, preproGHRH, and NPY proteins were down-regulated in them. The tissue results were highly corroborated with the serum data from the tumor patients and healthy women. Conclusions: The higher PC1 and CPE expressions as well as the transformation of more proGHRH into active GHRH peptide suggest stronger PC1/CPE-mediated neuropeptide processing in the tumor, whereas the lower PC2 expression as well as the transformation of less proNPY into active NPY peptide suggests a weak PC2-mediated processing in it. The alterations of the convertase expressions and processing show that there is a differential proprotein processing system in the tumor, which leads to the abnormal distributions of species, ratio, and concentration of (pro)peptide(s) in the microenvironment of cells. The latter may contribute to cancer progression.

[Objective] Phase I cells of Photorhabdus luminescens bacteria,symbiotically associated with entomopathogenic Heterorhabditis nematode,produce two types of intracellular crystalline inclusions,CipA and CipB,to support the symbiont.This study aimed to investigate the possible influence of Cip proteins on non-symbiotic Steinernema nematode.[Methods] Based on constructed Escherichia coli expression system of Cip proteins,co-culture system of recombinant E.coli bacteria and Steinernema sp.SY-5 nematode was set up.[Results] The Cip proteins significantly promoted the development of SY-5 nematode to make high adult rate of 65%-82%,gravid rate of 80%-95%,30-50 eggs per nematode,and low mortalities.[Conclusion] As the nutrient reserves for Heterorhabditis nematode,Cip proteins can be accepted and utilized by Steinernema nematode.The progress on these proteins will provide insights into the mechanisms governing bacteria-nematode symbiosis.

Objective To recombine human talin1 gene in Ecoli cells and purify the His-talin1 protein.Methods The talin1 cDNA was amplified by PCR.The talin1cDNA was inserted into the plasmid pET32a(+).By screening and sequencing the recombinant plasmid,the positive constructs were transformed into the E.coli BL21(DE3) host cells.After induced in IPTG,the recombinant products were purified in affinity chromatography and identified in SDS-PAGE and Western blot method.Results The 2.4 kb of human talin1 gene was obtained successfully.The recombinant plasmid pET32a(+)-talin1 was constructed.By analyzed in SDS-PAGE and Western blot methods,the fusion protein His-talin1 was purified successfully.Conclusion The prokaryotic expression system of His-talin1 was established.The His-talin1 protein purified provides to further study the relationship of talin1 and P-selectin.

A DNA clone containing α-galactosidase gene fragment(639 bp) was isolated from the constructed cDNA library of Absidia ramose WL511.The 5'-RACE and 3'-RACE techniques confirmed this gene's upstream and downstream sequences(1817 bp,1092 bp) Sequences analysis revealed that this gene with the full length of 2228 bp made a complete open reading frame of 2142 bp encoding a polypeptide of 713 amino acid residues with predicted molecular mass of 81 kDa and predicted pI of 5.2.This gene(GenBank No.DQ234280),belonging to α-galactosidase 36 family,showed a very low homology with other sources of α-galactosidase genes.The most similarity was 38 % between the α-galactosidases of Streptomyces avermitilis MA-4680 and A.ramose WL511.

Biochemistry is an important basic course in medical and pharmaceutical colleges.It is characterized by abstract theory,complex metabolism and interdisciplinary knowledge.This paper discusses the ideas,strategies and methods of biochemical theory teaching in three aspects: preparation before class,teaching in class and feedback after class.

cDNA sequence(GenBank No.DQ234280) of the α-galactosidase from Absidia ramose WL511 was cloned and inserted into the Pichia pastoris expression vector pPICZαA,and integrated into the genome of P.pastoris GS115 cells by electricity pulse.The culture supernatant showed 32U/ml of enzymatic activity at 30℃ with 0.5%(V/V) methanol added.The specific activity of purified recombinant enzyme at the grade of electrophoresis pure was 137 U/mg.The α-galactosidase expressed by P.pastoris was glycosyled protein with a 6kDa larger molecular mass than native enzyme,and a tetramer with the molecular weight of 348 kDa estimated by gel filtration and the subunit molecular weight of 87 kDa by SDS-PAGE.The pI value,optimal pH and temperature of the enzyme are 5.2,6.8 and 73 ℃.With pNPG as the substrate,the Km,Vmax,and kcat are 0.42mmol/Lol/L,413U/mg and 64 531 /min,respectively.This enzyme showed a stable activity at pH5.5 ～ 9.0 and below 60℃,still remained 54% of activity for 2 h at 75 ℃,and completely inactivated at 85℃.the secret-expressed α-galactosidase from A.ramose WL511 have better enzymatic properties of high thermostability,high specific activity and wide pH range.

Objective To sequence N-terminuses of the close two subunits of Agacutin,the separation of single subunit was completed for the sequencing.Methods The single subunits of Agacutin were obtained by using SDS-PAGE,subunit gel recovery and electrical transfer technique. The Edman degradation method was used for the sequencing.Results The subunit sequences of the 16 and 15 kDa were determined to be DCSSGWSSYEEHQYY and DSSGWSSYEGHEYYV,respectively.Conclusion According to above sequencing results,the NCBI Blast comparison showed that Agacutin is a novel thrombin-like enzyme.

AIM:To study the quality standard of Agacutin.Methods:By using the identification test,human plasma and bovine fibrinogen clotting activity,gel-filtration HPLC,SDS-PAGE,and Lachypleus amebocyte lysate,the quality standard of Agacutin was obtained.Results:The experimental results show 98% of average purity on HPLC,hetrodimer bands and whole molecular weight 31733±1157 D on SDS-PAGE.0.2 KU of Agacutin solution makes 0.2 ml of human plasma and 0.8 ml of bovine fibrinogen coagulating in 60±20 and 188±20 seconds.0.25 U of Agacutin showed a negative abnormal toxicity and allergic reaction.The dose 0.2 U/0.2 ml showed negative hemorrhagin reaction.1 KU of Agacutin product contained less than 10 EU of endotoxin.The experimental results also showed that Agacutin has no alkaline phosphatase,L-amino acid oxidase,phosphalipase-A activity.Conclusion According to the above results,the quality standard parameters,purity≥95%,molecular weight 31±2 kDa,endotoxin 10 EU/KU,human plasma and bovine fibrinogen clotting time 60±20 and 188 ± 20 seconds,abnormal toxicity and allergic test dose 0.25 U,and hemorrhagin test 0.2 U/0.2 ml,were determined.

虾青素是一种具有极强生物抗氧化性的酮式类胡萝卜素,在医药、食品、化妆品等方面有着极广阔的应用前景。目前生物技术法为虾青素的主要生产方法。本文以虾青素生产所涉及的关键酶、代谢途径及其改造为对象,系统综述了虾青素生产所涉及的代谢工程技术及其最新研究进展,并探讨了将来的发展前景。

Phase I cells of Photorhabdus luminescens produce two types of intracellular crystalline inclusion proteins designated CipA and CipB. The genes encoding CipA and CipB proteins from P. luminescens H06 were expressed respectively in Escherichia coli and these cells were used to feed the axenic first juveniles (J1) of three Steinernema nematode isolates in liquid cultures and on agar plates. In liquid cultures, the axenic J1 juveniles of all three test Steinernema nematode isolates were able to produce next dauer juveniles (DJs) in the E. coli cultures with at least one of the expressed Cip proteins, but unable to develop beyond the next J1 stage without expressed Cip proteins. For each target nematode isolate, addition of the supernatant of the bacterial culture of its Xenorhabdus symbiont to the tested liquid cultures did not induce the formation of DJs. However, on LB agar plates with different test E. coli cultures, all J1 juveniles of the three Steinernema strains finally developed into next DJs. It seemed that the metabolite pathway of the test bacteria in both culture systems was different. The presence of the Cip proteins has a significant influence on the DJ formation of the Steinernema nematodes in liquid culture system.

Phase I cells of Photorhabdus luminescens produce two types of intracellular crystalline inclusion proteins designated CipA and CipB. The genes encoding CipA and CipB proteins from P. luminescens H06 were expressed respectively in Escherichia coli and these cells were used to feed the axenic first juveniles (J1) of three Steinernema nematode isolates in liquid cultures and on agar plates. In liquid cultures, the axenic J1 juveniles of all three test Steinernema nematode isolates were able to produce next dauer juveniles (DJs) in the E. coli cultures with at least one of the expressed Cip proteins, but unable to develop beyond the next J1 stage without expressed Cip proteins. For each target nematode isolate, addition of the supernatant of the bacterial culture of its Xenorhabdus symbiont to the tested liquid cultures did not induce the formation of DJs. However, on LB agar plates with different test E. coli cultures, all J1 juveniles of the three Steinernema strains finally developed into next DJs. It seemed that the metabolite pathway of the test bacteria in both culture systems was different. The presence of the Cip has significant on the formation of the nematodes liquid culture nematodes in liquid and on plates.