BDNF-TAT融合蛋白在大肠杆菌中的表达纯化及体外活性测定
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摘要
神经退行性疾病,如老年性痴呆(Alzheimer's disease AD)、帕金森氏症(Parkinson's disease PD)、萎缩性侧索硬化(Amyotrophic lateral sclerosis, ALS)、和亨廷顿氏病(Huntington's disease HD)等,临床表现为肢体运动和学习记忆等功能障碍,主要原因是神经元功能的缺失或死亡。目前没有理想的治疗手段,在我国AD和PD这两种疾病已经累及大约1000万人给个人家庭和社会带来沉重负担。研究表明,神经营养因子家族成员(neurotrophic factors, NTFs)等,显示了很好的中枢神经系统神经退行性疾病的治疗前景。脑源性神经营养因子(BDNF)是神经营养因子家族成员包括之一,与其特异性受体酪氨酸激酶家族成员trkB结合,激活下游的信号传导途径,而发挥神经营养生物活性。
然而,血脑屏障(BBB)阻碍了大分子药物从血液中进入大脑发挥生理作用,人大脑血脑屏障(BBB)由血管内皮细胞紧密连接组成,BBB腔内面为血液,腔外面多为外膜细胞,星行细胞及少量的神经元。天然BDNF不能透过BBB进入大脑发挥神经营养活性。
蛋白质转导结构域(protein transduction domain, PTD),如来源于人类Ⅰ型免疫缺陷病毒的转录激活蛋白(Trans-activator transcription, TAT)的PTD,是一类富含正电荷能够将与其物理或化学连接的化合物、蛋白质、或核酸类分子,带过细胞膜进入细胞浆,胞核内,甚至BBB,发挥各自的生物学功能的结构域。PTD/TAT介导的蛋白质可穿过由脑血管内皮细胞组成的BBB,使所携带蛋白进入大脑发挥生理作用,而使一些在正常情况下是不能通过BBB的蛋白进入脑组织。
本实验中心前期通过基因工程的方法成功制备了编码成熟BDNF-TAT融合蛋白的基因,目的在于在世界上首先制备一种能通过BBB进入中枢神经系统,治疗神经退行性疾病的基因重组神经营养因子药物制剂。
基于前期研究,本文采用重组质粒PET-30(a)-BDNF-TAT转化至大肠杆菌BL21(DE3)plysS中优化表达,经阳离子交换树脂优化纯化及精氨酸倍比稀释法复性BDNF-TAT融合蛋白,尾静脉给药KM种小鼠后研究其脑靶向性及其在脑组织中的分布,体外培养SD大鼠新生一周鼠背根神经节神经元检测BDNF-TAT融合蛋白的生物学活性,为进一步研究可透过血脑屏障药物BDNF-TAT融合蛋白治疗神经退行性疾病提供物质基础。
目的
1.研究重组质粒PET-30(a)-BDNF-TAT转化至大肠杆菌BL21(DE3)plysS后BDNF-TAT融合蛋白的表达,并优化BDNF-TAT融合蛋白在大肠杆菌中表达及纯化的条件；
2.小鼠经尾静脉血管给予复性后BDNF-TAT融合蛋白,探讨其在小鼠脑组织中的脑靶向性及在小鼠脑组织中的分布；
3.体外培养新生大鼠背根神经节神经元细胞,进一步研究复性后BDNF-TAT融合蛋白体外促进神经元存活生长的活性。
方法
1.根据前期课题组经基因工程办法制备的重组质粒PET-30(a)-BDNF-TAT及,本文将该重组质粒转化至大肠杆菌BL21(DE3)plysS,37℃, 1.0mM IPTG诱导4h后低温离心(4℃,5000 rpm)收集菌体,超声裂解,高速离心后收集各步骤样品,通过15%SDS-PAGE和Western Blot分析确定融合蛋白BDNF-TAT在大肠杆菌中的表达形式；分别选取25℃、30℃、33℃、37℃、40℃为不同的诱导温度,对诱导温度进行优化；分别选取0.1mM、0.5mM、1.0mM、1.5mM为IPTG诱导浓度,对IPTG诱导浓度经行优化；选取1、2、4、6、8为诱导时间,对诱导时间经行优化；收集各条件下大肠杆菌并经行15%SDS-PAGE和(?)WesternBlot分析确定BDNF-TAT融合蛋白在大肠杆菌中的最佳表达条件。
2.重组质粒转化至大肠杆菌后,经25℃,0.5mM IPTG诱导4h后离心收集湿菌体,PBS重悬洗涤后用60%强度超声破碎仪裂解大肠杆菌,4℃、12000rpm低温高速高速离心去上清,沉淀用2M尿素和0.4%DOC洗涤并重复洗涤一次,低温高速离心去上清,包涵体沉淀用8M尿素4℃搅拌溶解14h,溶解后蛋白通过中高压层析系统泵入SP--Sepharose阳离子交换柱,选取pH7.0 NaCl浓度分别为0.1M和0.5M的洗脱液经行梯度洗脱优化不同离子强度对目的蛋白的洗脱能力；选取pH8.5,NaCl浓度为0.5M的洗脱液洗脱目的蛋白,对挂柱的目的蛋白经行分离纯化。洗脱后目的蛋白用0.4M精氨酸倍比稀释法除去目的蛋白中的尿素,同时复性融合蛋白BDNF-TAT。收集各步骤蛋白样品进行15%SDS-PAGE和Western Blot分析,确定最终过柱纯化优化方案。
3.随机取9只KM种小鼠作为实验对象,分为给药组,阴性对照组和空白对照组,每组3只,将纯化复性后的BDNF-TAT融合蛋白溶解于适量生理盐水,给药组动物尾静脉注射4μg BDNF-TAT,而阴性对照组给予等体积生理盐水,空白对照组不给药。4h后断头取闹组织,全蛋白提取试剂盒提取脑组织中全蛋白,免疫印迹法(Western Blot)鉴定脑组织中所含BDNF-TAT,对所有组中目的蛋白以p-actin作为内参进行上样量标准化后,以BDNF-TAT与β-actin灰度值之比为脑组织中BDNF-TAT相对含量,应用单向方差分析方法,比较给药组,阴性对照组贺空白组之间BDNF-TAT的相对含量是否有统计学意义。
4.随机取6只KM种小鼠作为实验对象,分为给药组和阴性对照组,每组3只,将纯化复性后的BDNF-TAT溶解于适量生理盐水,给药组动物尾静脉注射BDNF-TAT,而阴性对照组给予等体积生理盐水,4h后灌注4%多聚甲醛,断头取脑组织,小鼠脑组织4%多聚甲醛浸泡2h后浸入30%蔗糖溶液过夜,10μm厚度冰冻切片。切片用SABC免疫组织化学法鉴定BDNF-TAT在小鼠脑组织中的分布。
5.摘取新生一周左右SD大鼠背根神经节,经胶原酶Ⅳ和0.25%胰蛋白酶消化后用含10%FBS的DMEM培养基体外培养背根神经节神经元。将神经元细胞分为给药组,阳性对照组和空白对照组,给药组中DMEM培养基加入100ng/ml复性后融合蛋白BDNF-TAT,阳性对照组中DMEM培养基加入100ng/ml NGF,空白对照组中DMEM培养基不加入神经营养因子。培养基每2天换一次液,培养4天后用AChE神经元染色方法对神经元经行染色,观察神经元细胞生长情况。
结果
1.通过15%SDS-PAGE和Western Blot鉴定BDNF-TAT,大肠杆菌中表达BDNF-TAT融合蛋白在分子量约18kD处有表达,且在大肠杆菌裂解沉淀中目的蛋白较多,说明BDNF-TAT融合蛋白以包涵体沉淀形式存在；经不同温度,IPTG诱导浓度和诱导时间经行优化后,诱导温度在25℃,IPTG诱导浓度为0.5mmM诱导4h时BDNF-TAT融合蛋白在大肠杆菌中的表达量最高。
2.经15%SDS-PAGE和Western Blot分析,包涵体蛋白经2M尿素和0.4%DOC洗涤后大部分杂蛋白被洗涤干净,且8M尿素将大部分包涵体蛋白溶解,溶解后蛋白经SP--Sepharose阳离子交换柱纯化后,大部分目的蛋白被pH8.5、NaCl浓度为0.5 M的洗脱液洗脱下来,0.4M精氨酸复性后蛋白纯度约为90%,每升菌能产出约4.8 mg目的蛋白。
3. Western Blot鉴定脑组织中BDNF-TAT相对含量,对ECL发光显影胶片中给药组、阴性对照组和空白对照组中BDNF-TAT和β-actin的灰度扫描,以BDNF-TAT和p-actin的灰度比值作为各组中BDNF-TAT蛋白的相对表达量,结果以x±s表示,给药组中BDNF-TAT蛋白的相对表达量为1.897±0.286,阴性对照组中BDNF-TAT蛋白的相对表达量为0.615±0.234,空白对照组中BDNF-TAT蛋白的相对表达量为0.335±0.154。Levene检验方差齐性(P=0.447>0.05),方差齐。经单方向方差分析,各组间BDNF-TAT蛋白相对表达量有差异(F=39.019,P=0.000<0.05),差异有统计学意义。LSD法组间多重比较：给药组中BDNF-TAT相对表达量与阴性对照组中BDNF-TAT相对表达量相比(P=0.000<0.05),差异有统计学意义；给药组中BDNF-TAT相对表达量与空白对照组中BDNF-TAT相对表达量相比(P=0.000<0.05),差异有统计学意义；而阴性对照组中BDNF-TAT (?)目对表达量和空白对照组中BDNF-TAT相对表达量相比(P=0.188>0.05),差异没有统计学意义,说明BDNF-TAT通过外周给药能靶向至小鼠脑组织。
4.SABC免疫组织化学法鉴定BDNF-TAT经尾静脉给药KM种小鼠后其在脑组织中的分布,给药组中融合蛋白BDNF-TAT阳性染色明显较强,而生理盐水组中阳性染色明显弱于给药组,其阳性染色主要分布于海马区CA1、CA3和DG区。
AChE染色神经元后,空白对照组神经元细胞培养4天后胞体较小,细胞大部分死亡,突起较短。而给药组组和阳性对照组中神经元生长良好,突起较长,细胞数目明显较空白对照组多。每组孔中随机选取10个视野,经Image Pro Plus6.0软件测量细胞最长突起的长度,结果以x±s表示,结果给药组为143±13um,阳性对照组为154±13um,而空白对照组为64±15um, Levene检验方差齐性(F=0.039,P=0.961>0.05),方差齐。经单方向方差分析,各组间最长突起的长度有差异(F=126.523,P=0.000),差异有统计学意义。LSD法组间多重比较：给药组中最长突起长度与阳性性对照组中最长突起长度相比(P=0.087>0.05),差异没有统计学意义,而给药组中最长突起长度与空白对照组中最长突起长度相比(P=0.000<0.05),差异有统计学意义；阳性对照组中最长突起长度与空白对照组中最长突起长度相比(P=0.000<0.05),差异有统计学意义,说明BDNF-TAT在体外有能促进神经元突起生长。
通过Image Pro Plus6.0软件测量神经元胞体总面积,结果给药组为2686±242um2,阳性对照组为2864±169um2,而空白对照组为397±97um2。Levene检验方差齐性(F=0.039,P=0.047<0.05),方差不齐。经Welch分析,各组间神经元胞体总面积有差异(F=997.983,P=0.000),差异有统计学意义。Dunnett T3法组间多重比较：给药组中神经元胞体总面积与阳性性对照组中神经元胞体总面积相比(P=0.199>0.05),差异没有统计学意义,而给药组中神经元胞体总面积与空白对照组中神经元胞体总面积相比(P=0.000<0.05),差异有统计学意义；阳性对照组中神经元胞体总面积与空白对照组中神经元胞体总面积相比(P=0.000<0.05),差异有统计学意义,证明BDNF-TAT在体外有保护神经元生长和存活的生物学活性。
结论
1. BDNF-TAT融合蛋白能通过重组质粒PET-30(a)-BDNF-TAT转化至大肠杆菌BL21(DE3)plysS中以包涵体形式表达,分子量约为18kD左右。
2.包涵体蛋白经表达纯化优化并复性后,得到高纯度活性BDNF-TAT融合蛋白,每升菌产出目的蛋白约4.8mmg,纯度约为90%。
3.尾静脉注射复性后融合蛋白BDNF-TAT经Western Blot分析后,给药组小鼠脑组织中BDNF-TAT的相对含量较阴性对照组和空白对照组明显高(P>0.05),且阴性对照组与空白对照组中BDNF-TAT的相对含量无差异(P<0.05),证明复性后融合蛋白BDNF-TAT能通过外周尾静脉给药靶向至小鼠脑组织中。
4.SABC免疫组织化学法证明BDNF-TAT融合蛋白经外周尾静脉给药小鼠后,脑组织中BDNF-TAT主要分布在海马区CA1、CA3和DG区。
5.体外培养背根神经节神经元细胞表明,BDNF-TAT融合蛋白能促进神经元的生长存活。
Neurodegenerative diseases, such as Alzheimer's disease(AD), Parkinson's disease(PD), Amyotrophic lateral sclerosis(ALS) and Huntington's disease(HD), show movement disorders and learning or memory disorders in clinic, because of the death of neurons'in the brain.10 millions of people are suffered from AD and PD due to the absence of ideal therapy,which lead to heavy burden of Chinese people and society. A great deal of scientific research have proved that Neurotrophic Factors(NTFs) have a bright prospect in therapy of neurodegenerative diseases. Brain-derived neurotrophic factor(BDNF), one of NTFs, combines to its specific tyrosine kinase receptor trkB and then activates the downstream path and neurotrophy function.
But blood-brain barrier(BBB) can prevent macromolecular drugs from passing into the brain. BBB is formed by a lot of compact vascular endothelial cells. There are blood cells inside of BBB and outside of BBB is adventitial cells, astrocytes, neurons. The reason why nature BDNF can not get into brain to play neurotrophy activities is the BBB.
Protein transduction domain(PTD) is a structural domain containing rich positive charge, such as a kind of PTD from Trans-activator transcription(TAT) of HIV-1. Some compound, protein and nucleinic acid combined with PTD physically or chemically can pass through plasmalemma, nucleus even the BBB to play their activities. Some protein mediated by PTD can get through BBB and play its biologic activities, which can not pass into brain in normal.
In the previous researches, we have succeeded in encoding maturate recombinant BDNF-TAT fusion protein gene in our experimental center. In order to cure neurodegenerative diseases, we hope to produce a new recombinant gene pharmaceutical preparation for the first time, which can pass through the BBB in the world.
Based on the previous researches, the expression and purification of BDNF-TAT fusion protein in BL21(DE3)plysS E.coli were optimized in this study. The way of injection BDNF-TAT into caudal vein and promotion of neuron survival of BDNF-TAT fusion protein in vitro showed that BDNF-TAT can target into the brain and distribute in Hippocampal organization. This provided substantial material basis for further research about BDNF-TAT fusion protein in therapy of neurodegenerative diseases.
Objectives:
1.To optimize the protocol of expression and purification of BDNF-TAT fusion protein in the BL21(DE3)plysS E.coli.
2.To study the distribution of BDNF-TAT fusion protein in the brain tissue after being injected into caudal vein.
3.To study the neuron survival promoting effect of BDNF-TAT fusion protein in vitro.
Methods:
1.The recombinant vector PET-30(a)-BDNF-TAT was transfected into the BL21(DE3)plysS E.Coli,then the E.Coli was collected and suffered to ultrasonication. The E.Coli was inducted by the condition of 37℃, 1.0mM IPTG for 4 hours. All the samples were collected, then suffered to 15% SDS-PAGE and western blotting. Different induction temperatures (25℃,30℃,33℃,37℃,40℃), concentrations of IPTG (0.1 mM,0.5mM, 1.0mM,1.5mM), and induction time (1 hour,2 hours,4 hours,6 hours and 8hours) were tested for optimization. were also tested for optimization. The samples were collected and analyzed by 15% SDS-PAGE and Western Blotting to optimize the best induction condition of BDNF-TAT fusion protein. The recombinant vector PET-30(a)-BDNF-TAT was transformed into the BL21(DE3)plysS E.Coli and the the E.coli was colleted by high-speed centrifugation after being induced by the condition of 25℃with 0.5mM IPTG for 4 hours. Dispersed and washed by PBS, the E.coli was split in PBS by ultrasonication with the intensity of 60%. The supernatant was removed after centrifugating the splited E.coli by the condition of 12000rpm at 4℃. Precipitation was washed by 2M urea and 0.4% DOC by twice in turns. The supernatant was removed by low-temperature and high-speed centrifugation and the inclusion bodies was resolved by 8M urea with agitation for 14 hours at 4℃. The dissolved protein was isolated by SP-Sepharose cation exchange resin after pumped by mesohigh chromatographic system. To optimize the ionic strength of eluting of BDNF-TAT fusion protein,0.1 M and 0.5 M NaCl with pH 7.0 was used to wash the resin by the method of gradient elution. Finally, the washing way of 0.5 M NaCl with pH 8.5 occurred to isolate BDNF-TAT fusion protein. And then the interest protein was renatured by coubling dilution of 0.4 M Arginine to remove the urea. At last, we collected the samples above and analyzed the best purification conditions of BDNF-TAT fusion protein by 15% SDS-PAGE and Western Blot.
2.Nine Kunming rats were divided into 3 groups randomly:drug group, negative control group and blank cantrol group(n=3). The renatured fusion protein BDNF-TAT which was dispersed in proper saline was injected into rats by 4μg in drug group by caudal vein, and the rats of saline control group were injected with equal-volume saline while the rats of blank control group being treated with nothing. To identify the BDNF, the heads of rats were Cut off to fetch the brain tissues after treated with drug for 4 hours and the whole protein was extracted from the brain tissues by holoprotein extraction kit.β-actin was used to be the Inner-Reference.
3.Six Kunming rats was divided into 2 groups randomly:drug group and negative cantrol group(n=3). The renatured BDNF-TAT fusion protein were dispersed into proper saline and 4μg BDNF-TAT was injected into rats of drug group and equal-volume saline was injected into the rats of saline control group by caudal vein.All the heads of rats were cut off to fetch the brain tissues after treated by BDNF-TAT fusion protein for 4 hours. The tissues were soaked into 4% paraformaldehyde for 2 hours before being dipped into 30% sucrose for one night. The brain tissues freezed by -20℃were cut into the depth of lOum by freezing microtome. The distribution of BDNF-TAT in brain tissue was confirmed by the way of SABC immunohistochemisty.
4.The dorsal root ganglions of One-week old SD rats were separated and digested into single neuron cells by collagenase IV and 0.25% trypsin. The neuron cells were divided into 3 groups:drug group, positive control group and blank cantrol group. Drug group was treated by DMEM culture media mixed with 100ng/ml BDNF-TAT, while positive control group was treated by DMEM culture media mixed with 100ng/ml NGF and blank cantrol group was treated by no neurotrophic factors in DMEM culture media. Exchanged the media every two days and the neuron cells was stained by AChE methods. Observed the growth of the neuron cells.
Results:
1. A great quantity of BDNF-TAT fusion protein expressed in E.coli as inclusion bodies after transforming the recombinant vector PET-30(a)-BDNF-TAT into BL21(DE3)plysS with the Molecular Weight about 18kD by 15% SDS-PAGE and Western blotting. A lot of interest protein was found in precipitation after splited by ultrasonication and the results showed that BDNF-TAT fusion protein existed in E.coli as inclusion bodies. The condition of 25℃with 0.5mM IPTG for 4 hours was the best expression condition after optimization the three factors (induction temperature, concentration of IPTG, induction time).
2. After being analyzed by 15% SDS-PAGE and Western blotting,most impurities in inclusion bodies were washed clear by 2M urea and 0.4% DOC. And the inclusion bodies were resolved in 8 M urea before being purified by a SP-Sepharose cation exchange resin. Most BDNF-TAT fusion protein exsisted in the elution of 0.5M NaCl with pH 8.5. And about 4.8mg of BDNF-TAT fusion protein in one litre E.coli was collected with a purity of 90% after renatured by 0.4M Arginine.
3. The way of extracting the whole proteins from the brain tissue was conducted after tail vein injection of 4μg BDNF-TAT in Kunming mice for 3 hours.BDNF-TAT fusion protein targeted into the brain by the analysis of Western Bloting. The gray ratio between the gray ratio of BDNF-TAT and the gray ratio ofβ-actin stood the relative content of BDNF-TAT. The result (x±s) was:drug group 1.897±0.286, negative control group 0.615±0.234, blank control group 0.335±0.154. There is homogeneity of variance by Levene (P=0.447>0.05). There is significant difference of the longest lenth of axons between groups by One-way ANOVA (F=39.019, P=0.000). Multiple Comparisons by LSD:The relative content of BDNF-TAT in drug group was obvious higher than that in negative control group and blank control group (P<0.05) and there is no difference between negative control group and blank control group (P>0.05). It proved that BDNF-TAT can targeted into the brain of rat.
4. There were obvious dying areas in the brain of rats in drug group confirmed by SABC immunohistochemisty of BDNF-TAT fusion protein, which distributed in CA1,CA3 and DG of hippocampus. And there was no obvious dying areas in negtive control group.
Confirmed by AChE dying method, the results showed that the neuron cells grew better in drug group and positive group than that in blank group. Randomly chose 10 visual fields in each group to compare the longest lenth of axons of each group by Image Pro Plus 6.0. The result (x±s) was:drug groupl43±13μm,positive control group154±13μm,blank control group 64±15μm. There was homogeneity of variance by Levene (F=0.039,P=0.0.961>0.05). There was significant difference of the longest lenth of axons between groups by One-way ANOVA (F=126.523,P=0.000). Multiple Comparisions by LSD:There was no significant difference between drug group and positive control group (P=0.087>0.05).There was significant difference between drug group and blank control group (P=0.000<0.05). There was significant difference between positive control group and blank control group (P=0.000<0.05). Compared the total neuron cells area of each group by Image Pro Plus 6.0. The result (x±s) is:drug group 2686±242μm2, positive control group 2864±169μm2, blank control group 397±97μm2. There was no homogeneity of variance analysed by Levene (F=0.039, P=0.047<0.05). There was significant difference of the total neuron cells area between groups by analysed Welch (F=997.983, P=0.000). Multiple Comparisions by Dunnett T3:There was no significant difference between drug group and positive control group (P=0.199>0.05). There was significant difference between drug group and blank control group (P=0.000<0.05). There was significant difference between positive control group and blank control group (P=0.000<0.05). The results proved that BDNF-TAT fusion protein protected the neuron cells alive in vitro.
Conclusion:
1.The results implied that BDNF-TAT fusion protein could be expressed in E.coli as inclusion bodies after transforming the recombinant vector PET-30(a)-BDNF-TAT into BL21(DE3)plysS with the Molecular Weight about 18kD.
2.Inclusion bodies were purified by cation exchange resins and BDNF-TAT were renatured by 0.4M Arginine. About 4.8mg BDNF-TAT fusion protein was produced with the purity of 90% in one litre E.coli.
3.Western Bloting was used to analyze the relative contant of BDNF-TAT in the brain tissue of Kunming rats.The results showed that the relative content of BDNF-TAT in drug group was obvious higher than that in negative control group and blank control group(P<0.05). And there is no difference between negative control group and blank control group(P>0.05).It proved that BDNF-TAT targeted into the brain of rats.
4.The results showed that BDNF-TAT fusion protein distributes in the CA1,CA3 and DG of hippocampus that confirmed by SABC immunohistochemisty.
5.The neuron cells were cultivated with BDNF-TAT well.
然而,血脑屏障(BBB)阻碍了大分子药物从血液中进入大脑发挥生理作用,人大脑血脑屏障(BBB)由血管内皮细胞紧密连接组成,BBB腔内面为血液,腔外面多为外膜细胞,星行细胞及少量的神经元。天然BDNF不能透过BBB进入大脑发挥神经营养活性。
蛋白质转导结构域(protein transduction domain, PTD),如来源于人类Ⅰ型免疫缺陷病毒的转录激活蛋白(Trans-activator transcription, TAT)的PTD,是一类富含正电荷能够将与其物理或化学连接的化合物、蛋白质、或核酸类分子,带过细胞膜进入细胞浆,胞核内,甚至BBB,发挥各自的生物学功能的结构域。PTD/TAT介导的蛋白质可穿过由脑血管内皮细胞组成的BBB,使所携带蛋白进入大脑发挥生理作用,而使一些在正常情况下是不能通过BBB的蛋白进入脑组织。
本实验中心前期通过基因工程的方法成功制备了编码成熟BDNF-TAT融合蛋白的基因,目的在于在世界上首先制备一种能通过BBB进入中枢神经系统,治疗神经退行性疾病的基因重组神经营养因子药物制剂。
基于前期研究,本文采用重组质粒PET-30(a)-BDNF-TAT转化至大肠杆菌BL21(DE3)plysS中优化表达,经阳离子交换树脂优化纯化及精氨酸倍比稀释法复性BDNF-TAT融合蛋白,尾静脉给药KM种小鼠后研究其脑靶向性及其在脑组织中的分布,体外培养SD大鼠新生一周鼠背根神经节神经元检测BDNF-TAT融合蛋白的生物学活性,为进一步研究可透过血脑屏障药物BDNF-TAT融合蛋白治疗神经退行性疾病提供物质基础。
目的
1.研究重组质粒PET-30(a)-BDNF-TAT转化至大肠杆菌BL21(DE3)plysS后BDNF-TAT融合蛋白的表达,并优化BDNF-TAT融合蛋白在大肠杆菌中表达及纯化的条件；
2.小鼠经尾静脉血管给予复性后BDNF-TAT融合蛋白,探讨其在小鼠脑组织中的脑靶向性及在小鼠脑组织中的分布；
3.体外培养新生大鼠背根神经节神经元细胞,进一步研究复性后BDNF-TAT融合蛋白体外促进神经元存活生长的活性。
方法
1.根据前期课题组经基因工程办法制备的重组质粒PET-30(a)-BDNF-TAT及,本文将该重组质粒转化至大肠杆菌BL21(DE3)plysS,37℃, 1.0mM IPTG诱导4h后低温离心(4℃,5000 rpm)收集菌体,超声裂解,高速离心后收集各步骤样品,通过15%SDS-PAGE和Western Blot分析确定融合蛋白BDNF-TAT在大肠杆菌中的表达形式；分别选取25℃、30℃、33℃、37℃、40℃为不同的诱导温度,对诱导温度进行优化；分别选取0.1mM、0.5mM、1.0mM、1.5mM为IPTG诱导浓度,对IPTG诱导浓度经行优化；选取1、2、4、6、8为诱导时间,对诱导时间经行优化；收集各条件下大肠杆菌并经行15%SDS-PAGE和(?)WesternBlot分析确定BDNF-TAT融合蛋白在大肠杆菌中的最佳表达条件。
2.重组质粒转化至大肠杆菌后,经25℃,0.5mM IPTG诱导4h后离心收集湿菌体,PBS重悬洗涤后用60%强度超声破碎仪裂解大肠杆菌,4℃、12000rpm低温高速高速离心去上清,沉淀用2M尿素和0.4%DOC洗涤并重复洗涤一次,低温高速离心去上清,包涵体沉淀用8M尿素4℃搅拌溶解14h,溶解后蛋白通过中高压层析系统泵入SP--Sepharose阳离子交换柱,选取pH7.0 NaCl浓度分别为0.1M和0.5M的洗脱液经行梯度洗脱优化不同离子强度对目的蛋白的洗脱能力；选取pH8.5,NaCl浓度为0.5M的洗脱液洗脱目的蛋白,对挂柱的目的蛋白经行分离纯化。洗脱后目的蛋白用0.4M精氨酸倍比稀释法除去目的蛋白中的尿素,同时复性融合蛋白BDNF-TAT。收集各步骤蛋白样品进行15%SDS-PAGE和Western Blot分析,确定最终过柱纯化优化方案。
3.随机取9只KM种小鼠作为实验对象,分为给药组,阴性对照组和空白对照组,每组3只,将纯化复性后的BDNF-TAT融合蛋白溶解于适量生理盐水,给药组动物尾静脉注射4μg BDNF-TAT,而阴性对照组给予等体积生理盐水,空白对照组不给药。4h后断头取闹组织,全蛋白提取试剂盒提取脑组织中全蛋白,免疫印迹法(Western Blot)鉴定脑组织中所含BDNF-TAT,对所有组中目的蛋白以p-actin作为内参进行上样量标准化后,以BDNF-TAT与β-actin灰度值之比为脑组织中BDNF-TAT相对含量,应用单向方差分析方法,比较给药组,阴性对照组贺空白组之间BDNF-TAT的相对含量是否有统计学意义。
4.随机取6只KM种小鼠作为实验对象,分为给药组和阴性对照组,每组3只,将纯化复性后的BDNF-TAT溶解于适量生理盐水,给药组动物尾静脉注射BDNF-TAT,而阴性对照组给予等体积生理盐水,4h后灌注4%多聚甲醛,断头取脑组织,小鼠脑组织4%多聚甲醛浸泡2h后浸入30%蔗糖溶液过夜,10μm厚度冰冻切片。切片用SABC免疫组织化学法鉴定BDNF-TAT在小鼠脑组织中的分布。
5.摘取新生一周左右SD大鼠背根神经节,经胶原酶Ⅳ和0.25%胰蛋白酶消化后用含10%FBS的DMEM培养基体外培养背根神经节神经元。将神经元细胞分为给药组,阳性对照组和空白对照组,给药组中DMEM培养基加入100ng/ml复性后融合蛋白BDNF-TAT,阳性对照组中DMEM培养基加入100ng/ml NGF,空白对照组中DMEM培养基不加入神经营养因子。培养基每2天换一次液,培养4天后用AChE神经元染色方法对神经元经行染色,观察神经元细胞生长情况。
结果
1.通过15%SDS-PAGE和Western Blot鉴定BDNF-TAT,大肠杆菌中表达BDNF-TAT融合蛋白在分子量约18kD处有表达,且在大肠杆菌裂解沉淀中目的蛋白较多,说明BDNF-TAT融合蛋白以包涵体沉淀形式存在；经不同温度,IPTG诱导浓度和诱导时间经行优化后,诱导温度在25℃,IPTG诱导浓度为0.5mmM诱导4h时BDNF-TAT融合蛋白在大肠杆菌中的表达量最高。
2.经15%SDS-PAGE和Western Blot分析,包涵体蛋白经2M尿素和0.4%DOC洗涤后大部分杂蛋白被洗涤干净,且8M尿素将大部分包涵体蛋白溶解,溶解后蛋白经SP--Sepharose阳离子交换柱纯化后,大部分目的蛋白被pH8.5、NaCl浓度为0.5 M的洗脱液洗脱下来,0.4M精氨酸复性后蛋白纯度约为90%,每升菌能产出约4.8 mg目的蛋白。
3. Western Blot鉴定脑组织中BDNF-TAT相对含量,对ECL发光显影胶片中给药组、阴性对照组和空白对照组中BDNF-TAT和β-actin的灰度扫描,以BDNF-TAT和p-actin的灰度比值作为各组中BDNF-TAT蛋白的相对表达量,结果以x±s表示,给药组中BDNF-TAT蛋白的相对表达量为1.897±0.286,阴性对照组中BDNF-TAT蛋白的相对表达量为0.615±0.234,空白对照组中BDNF-TAT蛋白的相对表达量为0.335±0.154。Levene检验方差齐性(P=0.447>0.05),方差齐。经单方向方差分析,各组间BDNF-TAT蛋白相对表达量有差异(F=39.019,P=0.000<0.05),差异有统计学意义。LSD法组间多重比较：给药组中BDNF-TAT相对表达量与阴性对照组中BDNF-TAT相对表达量相比(P=0.000<0.05),差异有统计学意义；给药组中BDNF-TAT相对表达量与空白对照组中BDNF-TAT相对表达量相比(P=0.000<0.05),差异有统计学意义；而阴性对照组中BDNF-TAT (?)目对表达量和空白对照组中BDNF-TAT相对表达量相比(P=0.188>0.05),差异没有统计学意义,说明BDNF-TAT通过外周给药能靶向至小鼠脑组织。
4.SABC免疫组织化学法鉴定BDNF-TAT经尾静脉给药KM种小鼠后其在脑组织中的分布,给药组中融合蛋白BDNF-TAT阳性染色明显较强,而生理盐水组中阳性染色明显弱于给药组,其阳性染色主要分布于海马区CA1、CA3和DG区。
AChE染色神经元后,空白对照组神经元细胞培养4天后胞体较小,细胞大部分死亡,突起较短。而给药组组和阳性对照组中神经元生长良好,突起较长,细胞数目明显较空白对照组多。每组孔中随机选取10个视野,经Image Pro Plus6.0软件测量细胞最长突起的长度,结果以x±s表示,结果给药组为143±13um,阳性对照组为154±13um,而空白对照组为64±15um, Levene检验方差齐性(F=0.039,P=0.961>0.05),方差齐。经单方向方差分析,各组间最长突起的长度有差异(F=126.523,P=0.000),差异有统计学意义。LSD法组间多重比较：给药组中最长突起长度与阳性性对照组中最长突起长度相比(P=0.087>0.05),差异没有统计学意义,而给药组中最长突起长度与空白对照组中最长突起长度相比(P=0.000<0.05),差异有统计学意义；阳性对照组中最长突起长度与空白对照组中最长突起长度相比(P=0.000<0.05),差异有统计学意义,说明BDNF-TAT在体外有能促进神经元突起生长。
通过Image Pro Plus6.0软件测量神经元胞体总面积,结果给药组为2686±242um2,阳性对照组为2864±169um2,而空白对照组为397±97um2。Levene检验方差齐性(F=0.039,P=0.047<0.05),方差不齐。经Welch分析,各组间神经元胞体总面积有差异(F=997.983,P=0.000),差异有统计学意义。Dunnett T3法组间多重比较：给药组中神经元胞体总面积与阳性性对照组中神经元胞体总面积相比(P=0.199>0.05),差异没有统计学意义,而给药组中神经元胞体总面积与空白对照组中神经元胞体总面积相比(P=0.000<0.05),差异有统计学意义；阳性对照组中神经元胞体总面积与空白对照组中神经元胞体总面积相比(P=0.000<0.05),差异有统计学意义,证明BDNF-TAT在体外有保护神经元生长和存活的生物学活性。
结论
1. BDNF-TAT融合蛋白能通过重组质粒PET-30(a)-BDNF-TAT转化至大肠杆菌BL21(DE3)plysS中以包涵体形式表达,分子量约为18kD左右。
2.包涵体蛋白经表达纯化优化并复性后,得到高纯度活性BDNF-TAT融合蛋白,每升菌产出目的蛋白约4.8mmg,纯度约为90%。
3.尾静脉注射复性后融合蛋白BDNF-TAT经Western Blot分析后,给药组小鼠脑组织中BDNF-TAT的相对含量较阴性对照组和空白对照组明显高(P>0.05),且阴性对照组与空白对照组中BDNF-TAT的相对含量无差异(P<0.05),证明复性后融合蛋白BDNF-TAT能通过外周尾静脉给药靶向至小鼠脑组织中。
4.SABC免疫组织化学法证明BDNF-TAT融合蛋白经外周尾静脉给药小鼠后,脑组织中BDNF-TAT主要分布在海马区CA1、CA3和DG区。
5.体外培养背根神经节神经元细胞表明,BDNF-TAT融合蛋白能促进神经元的生长存活。
Neurodegenerative diseases, such as Alzheimer's disease(AD), Parkinson's disease(PD), Amyotrophic lateral sclerosis(ALS) and Huntington's disease(HD), show movement disorders and learning or memory disorders in clinic, because of the death of neurons'in the brain.10 millions of people are suffered from AD and PD due to the absence of ideal therapy,which lead to heavy burden of Chinese people and society. A great deal of scientific research have proved that Neurotrophic Factors(NTFs) have a bright prospect in therapy of neurodegenerative diseases. Brain-derived neurotrophic factor(BDNF), one of NTFs, combines to its specific tyrosine kinase receptor trkB and then activates the downstream path and neurotrophy function.
But blood-brain barrier(BBB) can prevent macromolecular drugs from passing into the brain. BBB is formed by a lot of compact vascular endothelial cells. There are blood cells inside of BBB and outside of BBB is adventitial cells, astrocytes, neurons. The reason why nature BDNF can not get into brain to play neurotrophy activities is the BBB.
Protein transduction domain(PTD) is a structural domain containing rich positive charge, such as a kind of PTD from Trans-activator transcription(TAT) of HIV-1. Some compound, protein and nucleinic acid combined with PTD physically or chemically can pass through plasmalemma, nucleus even the BBB to play their activities. Some protein mediated by PTD can get through BBB and play its biologic activities, which can not pass into brain in normal.
In the previous researches, we have succeeded in encoding maturate recombinant BDNF-TAT fusion protein gene in our experimental center. In order to cure neurodegenerative diseases, we hope to produce a new recombinant gene pharmaceutical preparation for the first time, which can pass through the BBB in the world.
Based on the previous researches, the expression and purification of BDNF-TAT fusion protein in BL21(DE3)plysS E.coli were optimized in this study. The way of injection BDNF-TAT into caudal vein and promotion of neuron survival of BDNF-TAT fusion protein in vitro showed that BDNF-TAT can target into the brain and distribute in Hippocampal organization. This provided substantial material basis for further research about BDNF-TAT fusion protein in therapy of neurodegenerative diseases.
Objectives:
1.To optimize the protocol of expression and purification of BDNF-TAT fusion protein in the BL21(DE3)plysS E.coli.
2.To study the distribution of BDNF-TAT fusion protein in the brain tissue after being injected into caudal vein.
3.To study the neuron survival promoting effect of BDNF-TAT fusion protein in vitro.
Methods:
1.The recombinant vector PET-30(a)-BDNF-TAT was transfected into the BL21(DE3)plysS E.Coli,then the E.Coli was collected and suffered to ultrasonication. The E.Coli was inducted by the condition of 37℃, 1.0mM IPTG for 4 hours. All the samples were collected, then suffered to 15% SDS-PAGE and western blotting. Different induction temperatures (25℃,30℃,33℃,37℃,40℃), concentrations of IPTG (0.1 mM,0.5mM, 1.0mM,1.5mM), and induction time (1 hour,2 hours,4 hours,6 hours and 8hours) were tested for optimization. were also tested for optimization. The samples were collected and analyzed by 15% SDS-PAGE and Western Blotting to optimize the best induction condition of BDNF-TAT fusion protein. The recombinant vector PET-30(a)-BDNF-TAT was transformed into the BL21(DE3)plysS E.Coli and the the E.coli was colleted by high-speed centrifugation after being induced by the condition of 25℃with 0.5mM IPTG for 4 hours. Dispersed and washed by PBS, the E.coli was split in PBS by ultrasonication with the intensity of 60%. The supernatant was removed after centrifugating the splited E.coli by the condition of 12000rpm at 4℃. Precipitation was washed by 2M urea and 0.4% DOC by twice in turns. The supernatant was removed by low-temperature and high-speed centrifugation and the inclusion bodies was resolved by 8M urea with agitation for 14 hours at 4℃. The dissolved protein was isolated by SP-Sepharose cation exchange resin after pumped by mesohigh chromatographic system. To optimize the ionic strength of eluting of BDNF-TAT fusion protein,0.1 M and 0.5 M NaCl with pH 7.0 was used to wash the resin by the method of gradient elution. Finally, the washing way of 0.5 M NaCl with pH 8.5 occurred to isolate BDNF-TAT fusion protein. And then the interest protein was renatured by coubling dilution of 0.4 M Arginine to remove the urea. At last, we collected the samples above and analyzed the best purification conditions of BDNF-TAT fusion protein by 15% SDS-PAGE and Western Blot.
2.Nine Kunming rats were divided into 3 groups randomly:drug group, negative control group and blank cantrol group(n=3). The renatured fusion protein BDNF-TAT which was dispersed in proper saline was injected into rats by 4μg in drug group by caudal vein, and the rats of saline control group were injected with equal-volume saline while the rats of blank control group being treated with nothing. To identify the BDNF, the heads of rats were Cut off to fetch the brain tissues after treated with drug for 4 hours and the whole protein was extracted from the brain tissues by holoprotein extraction kit.β-actin was used to be the Inner-Reference.
3.Six Kunming rats was divided into 2 groups randomly:drug group and negative cantrol group(n=3). The renatured BDNF-TAT fusion protein were dispersed into proper saline and 4μg BDNF-TAT was injected into rats of drug group and equal-volume saline was injected into the rats of saline control group by caudal vein.All the heads of rats were cut off to fetch the brain tissues after treated by BDNF-TAT fusion protein for 4 hours. The tissues were soaked into 4% paraformaldehyde for 2 hours before being dipped into 30% sucrose for one night. The brain tissues freezed by -20℃were cut into the depth of lOum by freezing microtome. The distribution of BDNF-TAT in brain tissue was confirmed by the way of SABC immunohistochemisty.
4.The dorsal root ganglions of One-week old SD rats were separated and digested into single neuron cells by collagenase IV and 0.25% trypsin. The neuron cells were divided into 3 groups:drug group, positive control group and blank cantrol group. Drug group was treated by DMEM culture media mixed with 100ng/ml BDNF-TAT, while positive control group was treated by DMEM culture media mixed with 100ng/ml NGF and blank cantrol group was treated by no neurotrophic factors in DMEM culture media. Exchanged the media every two days and the neuron cells was stained by AChE methods. Observed the growth of the neuron cells.
Results:
1. A great quantity of BDNF-TAT fusion protein expressed in E.coli as inclusion bodies after transforming the recombinant vector PET-30(a)-BDNF-TAT into BL21(DE3)plysS with the Molecular Weight about 18kD by 15% SDS-PAGE and Western blotting. A lot of interest protein was found in precipitation after splited by ultrasonication and the results showed that BDNF-TAT fusion protein existed in E.coli as inclusion bodies. The condition of 25℃with 0.5mM IPTG for 4 hours was the best expression condition after optimization the three factors (induction temperature, concentration of IPTG, induction time).
2. After being analyzed by 15% SDS-PAGE and Western blotting,most impurities in inclusion bodies were washed clear by 2M urea and 0.4% DOC. And the inclusion bodies were resolved in 8 M urea before being purified by a SP-Sepharose cation exchange resin. Most BDNF-TAT fusion protein exsisted in the elution of 0.5M NaCl with pH 8.5. And about 4.8mg of BDNF-TAT fusion protein in one litre E.coli was collected with a purity of 90% after renatured by 0.4M Arginine.
3. The way of extracting the whole proteins from the brain tissue was conducted after tail vein injection of 4μg BDNF-TAT in Kunming mice for 3 hours.BDNF-TAT fusion protein targeted into the brain by the analysis of Western Bloting. The gray ratio between the gray ratio of BDNF-TAT and the gray ratio ofβ-actin stood the relative content of BDNF-TAT. The result (x±s) was:drug group 1.897±0.286, negative control group 0.615±0.234, blank control group 0.335±0.154. There is homogeneity of variance by Levene (P=0.447>0.05). There is significant difference of the longest lenth of axons between groups by One-way ANOVA (F=39.019, P=0.000). Multiple Comparisons by LSD:The relative content of BDNF-TAT in drug group was obvious higher than that in negative control group and blank control group (P<0.05) and there is no difference between negative control group and blank control group (P>0.05). It proved that BDNF-TAT can targeted into the brain of rat.
4. There were obvious dying areas in the brain of rats in drug group confirmed by SABC immunohistochemisty of BDNF-TAT fusion protein, which distributed in CA1,CA3 and DG of hippocampus. And there was no obvious dying areas in negtive control group.
Confirmed by AChE dying method, the results showed that the neuron cells grew better in drug group and positive group than that in blank group. Randomly chose 10 visual fields in each group to compare the longest lenth of axons of each group by Image Pro Plus 6.0. The result (x±s) was:drug groupl43±13μm,positive control group154±13μm,blank control group 64±15μm. There was homogeneity of variance by Levene (F=0.039,P=0.0.961>0.05). There was significant difference of the longest lenth of axons between groups by One-way ANOVA (F=126.523,P=0.000). Multiple Comparisions by LSD:There was no significant difference between drug group and positive control group (P=0.087>0.05).There was significant difference between drug group and blank control group (P=0.000<0.05). There was significant difference between positive control group and blank control group (P=0.000<0.05). Compared the total neuron cells area of each group by Image Pro Plus 6.0. The result (x±s) is:drug group 2686±242μm2, positive control group 2864±169μm2, blank control group 397±97μm2. There was no homogeneity of variance analysed by Levene (F=0.039, P=0.047<0.05). There was significant difference of the total neuron cells area between groups by analysed Welch (F=997.983, P=0.000). Multiple Comparisions by Dunnett T3:There was no significant difference between drug group and positive control group (P=0.199>0.05). There was significant difference between drug group and blank control group (P=0.000<0.05). There was significant difference between positive control group and blank control group (P=0.000<0.05). The results proved that BDNF-TAT fusion protein protected the neuron cells alive in vitro.
Conclusion:
1.The results implied that BDNF-TAT fusion protein could be expressed in E.coli as inclusion bodies after transforming the recombinant vector PET-30(a)-BDNF-TAT into BL21(DE3)plysS with the Molecular Weight about 18kD.
2.Inclusion bodies were purified by cation exchange resins and BDNF-TAT were renatured by 0.4M Arginine. About 4.8mg BDNF-TAT fusion protein was produced with the purity of 90% in one litre E.coli.
3.Western Bloting was used to analyze the relative contant of BDNF-TAT in the brain tissue of Kunming rats.The results showed that the relative content of BDNF-TAT in drug group was obvious higher than that in negative control group and blank control group(P<0.05). And there is no difference between negative control group and blank control group(P>0.05).It proved that BDNF-TAT targeted into the brain of rats.
4.The results showed that BDNF-TAT fusion protein distributes in the CA1,CA3 and DG of hippocampus that confirmed by SABC immunohistochemisty.
5.The neuron cells were cultivated with BDNF-TAT well.
引文
[1]疼痛、药物成瘾和神经退行性疾病最新研究进展JOURNAL OF PEKING UNIVERSITY(HEALTH SCIENCES) Vol41 No.3 Jun.2009:249-253
[2]Lu B, Pang PT and Woo NH. The yin and yang of neurotrophin action. Nature Reviews Neuroscience.2005;6:603-614
[3]Lee R, Kermani P, Teng KK, et al. Regulation of cell survival by secreted proneurotrophins. Science.2001;294:1945-1948.
[4]Tago H Kimura H and Maeda T. Visualization of detailed ace. tylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure. J Histochem cytochem 1986; 34(11):1431-1438
[5]Pardridge WM. Brain Drug Targeting:the future of Brain Drug Development.Cambridge Univ.Press, Cambridge,2001.
[6]Asoh S, et al. Protection against ischemic brain injury by protein therapeutics. Proc. Natl. Acad. Sci. USA.2002; 99:17107-17112
[7]Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses[J]. Nature ReviewsDrug Discovery,2002,1:131-139.
[8]Wadia JS,et al.Transducible TAT-HA fusogenic peptides enhances escape TAT-fusion proteins after lipid raft macropinocytosis. Nature Medicine. 2004; 10(3):310-315.
[9]Hans Peter Soensen, Kim Kusk Mortensen.Advanced genetic strategies for recombinant protein expression in Escherichia coli.Journal of Biotechnology 115(2005)113-128.
[10]Lyn-Marie Birkholtzl, Gregory Blatch2, Theresa L Coetzer3,Heinrich C Hoppel,4, Esmare Humanl, Elizabeth J Morrisl,5, Zoleka Ngcetel, Lyndon Oldfield4, Robyn Roth4, Addmore Shonhai6, Linda Stephens2 and Abraham I Louw.Heterologous expression of plasmodial proteins for structural studies and functional annotation.Malaria Journal 2008,7:197.
[11]TAN Hongyi, PAN Pinhua, HU Chengping.Primary culture method to obtain high purified dorsal root ganglion neurons of rats in vitro.International Journal of Pathology and Clinical Medicine.2009 No.3 Vol.29.
[12]Huang EJ and Reichardt LF。Trk receptors:roles in neuronal signal transduction. Annu. Rev. Biochem.2003; 72:609-642
[13]Downward J.PI 3-kinase, Akt and cell survival. Semin. Cell Dev. Biol.2004; 15:177-182.
[14]王晓娟,郭力,等。GDNF促进大鼠背根神经元的存活和突起生长Basic Medjeal sciences and clinics,2003;06-0625-05
[15]DING Hui,HE Ping.Progress in study on BDNF and its appl ication in pediatrics国外医学妇幼保健分.2005；16(1)13-15
[16]Young-Min Kim & Doman Kim.Characterization of novel thermostable dextranase from Thermotoga lettingae TMO.Appl Microbiol Biotechnol (2010) 85:581-587
[17]Moon-Soo Kim, Jiao Song,etl.Determining protein stability in cell lysates by pulse proteolysis and Western blotting.PROTEIN SCIENCE 2009 VOL 18:1051-1059
[18]Pedro Castanheira, Susana Moreira,etl.Escherichia coli expression, refolding and characterization of human laforin.Protein Expression and Purification 71 (2010) 195-199
[19]Elavazhagan Murugan, Rong Kong,etl.Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa.Protein Expression and Purification 71 (2010) 132-138
[20]Marcelo F. Marcondes, Ricardo J.S. Torquato,etl.Mitochondrial intermediate peptidase:Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates.Biochemical and Biophysical Research Communications 391 (2010) 123-128
[21]SMITA RAGHAVA, BIPASHA BARUA,etl.Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies.Protein Science (2008),17:1987-1997.
[22]Xianglei Gao,Wei Chen,etl.Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion. Appl Microbiol Biotechnol (2010) 85:1051-1060
[23]Danielle L Graham, Scott Edwards,etl.Dynamic BDNF activity in nucleus accumbens with cocaine use increases self-administration and relapse. NATURE NEUROSCIENCE VOLUME 10 NUMBER 8 AUGUST 2007
[24]Hao Zhua,Wen-JinWang,etl.Effect of panaxydol on hypoxia-induced cell death and expression and secretion of neurotrophic factors (NTFs) in hypoxic primary cultured Schwann cells.Chemico-Biological Interactions 174 (2008) 44-50
[25]Woo Hyun Park,M.D.,etl.Acetylcholinesterase Histochemistry of Rectal Suxtio Biopsies in the Diagnosis of Hisrschsprung's Disease. Journal of Korean Medical Science.(1992)7,4:353-359
[1]于文国,陶秀娥.包涵体蛋白复性及其影响因素[J]河北工业科技,2007,(05).
[2]宁云山,李妍,王小宁包含体蛋白质的复性研究进展.生物技术通讯,2001,12(3)237-248
[3]王进,华子春,方勇,唐卫东,朱德煦,冯若,朱昌平,黄金兰,陈兆华.功率超声法对蛋白质包涵体的解聚.生物化学和生物物理进展,1995,22(6)：543-545
[4]耿信笃.高效疏水色谱在蛋白质复性中应用.科学通报1994,44(19)：2046-2049
[5]万雪,王磊,宁官保.包涵体及其复性研究概况[J].畜牧兽医科技信息,2005,(02)
[6]井明艳,孙建义.蛋白质的折叠调控与包涵体的形成[J].浙江大学学报(农业与生命科学版),2004,(06)
[7]方敏,黄华棵.包涵体蛋白体外复性的研究进展[J]生物工程学报,2001,(06).[8]冯小黎.重组包涵体蛋白质的折叠复性[J]生物化学与生物物理进展,2001,(04).
[8]凌明圣,许祥裕,丁树标.以包涵体形式存在的重组蛋白的纯化和体外折叠[J]中国生化药物杂志,1995,(03).
[9]杨晓仪,林键,吴文言.重组蛋白包涵体的复性研究[J]生命科学研究,2004,(02).
[10]罗惠霞,李敏,王玉炯.包涵体蛋白复性的几种方法[J].生物技术通报,2007,(05).
[11]高飞,范清林,邹文艺,宋礼华.包涵体蛋白的层析复性技术研究进展[J].生物技术通报,2006,(02).
[12]艾恒,莫书荣,鲁波.包涵体研究进展[J].中国医学文摘.内科学,2006,(02).
[13]邹平.重组包涵体蛋白质复性[J].厦门科技,2005,(05).
[14]吉清,何凤田.包涵体复性的研究进展[J].国外医学.临床生物化学与检验学分册,2004,(06).
[2]Lu B, Pang PT and Woo NH. The yin and yang of neurotrophin action. Nature Reviews Neuroscience.2005;6:603-614
[3]Lee R, Kermani P, Teng KK, et al. Regulation of cell survival by secreted proneurotrophins. Science.2001;294:1945-1948.
[4]Tago H Kimura H and Maeda T. Visualization of detailed ace. tylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure. J Histochem cytochem 1986; 34(11):1431-1438
[5]Pardridge WM. Brain Drug Targeting:the future of Brain Drug Development.Cambridge Univ.Press, Cambridge,2001.
[6]Asoh S, et al. Protection against ischemic brain injury by protein therapeutics. Proc. Natl. Acad. Sci. USA.2002; 99:17107-17112
[7]Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses[J]. Nature ReviewsDrug Discovery,2002,1:131-139.
[8]Wadia JS,et al.Transducible TAT-HA fusogenic peptides enhances escape TAT-fusion proteins after lipid raft macropinocytosis. Nature Medicine. 2004; 10(3):310-315.
[9]Hans Peter Soensen, Kim Kusk Mortensen.Advanced genetic strategies for recombinant protein expression in Escherichia coli.Journal of Biotechnology 115(2005)113-128.
[10]Lyn-Marie Birkholtzl, Gregory Blatch2, Theresa L Coetzer3,Heinrich C Hoppel,4, Esmare Humanl, Elizabeth J Morrisl,5, Zoleka Ngcetel, Lyndon Oldfield4, Robyn Roth4, Addmore Shonhai6, Linda Stephens2 and Abraham I Louw.Heterologous expression of plasmodial proteins for structural studies and functional annotation.Malaria Journal 2008,7:197.
[11]TAN Hongyi, PAN Pinhua, HU Chengping.Primary culture method to obtain high purified dorsal root ganglion neurons of rats in vitro.International Journal of Pathology and Clinical Medicine.2009 No.3 Vol.29.
[12]Huang EJ and Reichardt LF。Trk receptors:roles in neuronal signal transduction. Annu. Rev. Biochem.2003; 72:609-642
[13]Downward J.PI 3-kinase, Akt and cell survival. Semin. Cell Dev. Biol.2004; 15:177-182.
[14]王晓娟,郭力,等。GDNF促进大鼠背根神经元的存活和突起生长Basic Medjeal sciences and clinics,2003;06-0625-05
[15]DING Hui,HE Ping.Progress in study on BDNF and its appl ication in pediatrics国外医学妇幼保健分.2005；16(1)13-15
[16]Young-Min Kim & Doman Kim.Characterization of novel thermostable dextranase from Thermotoga lettingae TMO.Appl Microbiol Biotechnol (2010) 85:581-587
[17]Moon-Soo Kim, Jiao Song,etl.Determining protein stability in cell lysates by pulse proteolysis and Western blotting.PROTEIN SCIENCE 2009 VOL 18:1051-1059
[18]Pedro Castanheira, Susana Moreira,etl.Escherichia coli expression, refolding and characterization of human laforin.Protein Expression and Purification 71 (2010) 195-199
[19]Elavazhagan Murugan, Rong Kong,etl.Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa.Protein Expression and Purification 71 (2010) 132-138
[20]Marcelo F. Marcondes, Ricardo J.S. Torquato,etl.Mitochondrial intermediate peptidase:Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates.Biochemical and Biophysical Research Communications 391 (2010) 123-128
[21]SMITA RAGHAVA, BIPASHA BARUA,etl.Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies.Protein Science (2008),17:1987-1997.
[22]Xianglei Gao,Wei Chen,etl.Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion. Appl Microbiol Biotechnol (2010) 85:1051-1060
[23]Danielle L Graham, Scott Edwards,etl.Dynamic BDNF activity in nucleus accumbens with cocaine use increases self-administration and relapse. NATURE NEUROSCIENCE VOLUME 10 NUMBER 8 AUGUST 2007
[24]Hao Zhua,Wen-JinWang,etl.Effect of panaxydol on hypoxia-induced cell death and expression and secretion of neurotrophic factors (NTFs) in hypoxic primary cultured Schwann cells.Chemico-Biological Interactions 174 (2008) 44-50
[25]Woo Hyun Park,M.D.,etl.Acetylcholinesterase Histochemistry of Rectal Suxtio Biopsies in the Diagnosis of Hisrschsprung's Disease. Journal of Korean Medical Science.(1992)7,4:353-359
[1]于文国,陶秀娥.包涵体蛋白复性及其影响因素[J]河北工业科技,2007,(05).
[2]宁云山,李妍,王小宁包含体蛋白质的复性研究进展.生物技术通讯,2001,12(3)237-248
[3]王进,华子春,方勇,唐卫东,朱德煦,冯若,朱昌平,黄金兰,陈兆华.功率超声法对蛋白质包涵体的解聚.生物化学和生物物理进展,1995,22(6)：543-545
[4]耿信笃.高效疏水色谱在蛋白质复性中应用.科学通报1994,44(19)：2046-2049
[5]万雪,王磊,宁官保.包涵体及其复性研究概况[J].畜牧兽医科技信息,2005,(02)
[6]井明艳,孙建义.蛋白质的折叠调控与包涵体的形成[J].浙江大学学报(农业与生命科学版),2004,(06)
[7]方敏,黄华棵.包涵体蛋白体外复性的研究进展[J]生物工程学报,2001,(06).[8]冯小黎.重组包涵体蛋白质的折叠复性[J]生物化学与生物物理进展,2001,(04).
[8]凌明圣,许祥裕,丁树标.以包涵体形式存在的重组蛋白的纯化和体外折叠[J]中国生化药物杂志,1995,(03).
[9]杨晓仪,林键,吴文言.重组蛋白包涵体的复性研究[J]生命科学研究,2004,(02).
[10]罗惠霞,李敏,王玉炯.包涵体蛋白复性的几种方法[J].生物技术通报,2007,(05).
[11]高飞,范清林,邹文艺,宋礼华.包涵体蛋白的层析复性技术研究进展[J].生物技术通报,2006,(02).
[12]艾恒,莫书荣,鲁波.包涵体研究进展[J].中国医学文摘.内科学,2006,(02).
[13]邹平.重组包涵体蛋白质复性[J].厦门科技,2005,(05).
[14]吉清,何凤田.包涵体复性的研究进展[J].国外医学.临床生物化学与检验学分册,2004,(06).