巴斯德毕赤酵母表达的突变型人白细胞介素-2的发酵条件与纯化研究

在以前的研究中,通过蛋白质工程技术获得了三突变体白细胞介素_2基因(编码125位半胱氨酸→丙氨酸;18位亮氨酸→蛋氨酸;19位亮氨酸→丝氨酸) ,并在毕赤酵母中加以表达。进一步优化表达条件,其最适诱导条件:80 %以上的通气,诱导2d ,初始pH60 ,甲醇终浓度为10%。在上述条件下表达量占菌体总蛋白的30%以上,大约200mg L。建立了一套从毕赤酵母表达上清中分离纯化分泌型表达蛋白IL_2的方法,经离心,超滤浓缩,强阳离子交换S柱和分子筛层析得到纯化的突变型和野生型IL-2 ;其得率为27% ,纯度达电泳纯并且HPLC检测只有一个峰。纯化的突变蛋白对CTLL-2细胞具有刺激性;与野生型IL-2相比,在各种温度条件下储存的突变蛋白保留有更高的活性;突变型IL-2的活力是野生型的4～5倍,具有更高的利用价值。     

重组人巨细胞病毒嵌合肽基因在毕赤酵母中的克隆和表达

为了在毕赤酵母中表达带有组氨酸纯化标记的重组人巨细胞病毒嵌合肽(rHCMVp),根据 其基因序列,设计引物从pPIC9K2rHCMVp 上扩增得到目的基因片段,并导入毕赤酵母诱导型表达 载体pPICZαA 中。通过电击将线性化的重组质粒转化到毕赤酵母X33 细胞中,筛选获得表达量 较高的重组菌株,研究了该菌株生长的培养条件,包括不同诱导时间、甲醇浓度、pH 值对人巨细 胞病毒嵌合肽表达的影响。2L 发酵罐进行了高密度发酵,经1 %的甲醇、pH610 的条件下诱导 48h,最终菌体密度OD600达到180,每升发酵液中含目的蛋白7817mg,产量比摇瓶提高了418 倍。 rHCMVp 可通过高密度发酵大量获得。     

猪IL-18在毕赤酵母中的表达及活性分析

采用PCR方法从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18,电激法转化入毕赤酵母GS115,采用G418抗性梯度法筛选得到多拷贝重组菌株,甲醇诱导表达,应用SDS-PAGE分析重组蛋白的表达情况,并将表达蛋白用凝胶层析柱纯化后,用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18,其表达在72h时达高峰,分泌量可达160mg/L,纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性,说明本试验已在毕赤酵母中在国内首次成功表达了具有生物学活性的pIL-18。     

重组酵母产人IFN-α2a与LL-37融合蛋白的工艺优化

通过单因素试验探索重组酵母P.pastoris GS115LI产人LL-37基因与IFN-α2a融合蛋白的最佳条件。结果显示,重组酵母菌产蛋白酶的最佳表达条件是利用BMMY为诱导培养基,以接种量OD600=5.5、温度26℃、p H6.0、诱导剂(甲醇)为每隔24 h添加1.0%、振荡速度为200 r/min条件下连续诱导144 h。在最佳培养条件下,发酵液中的LL-37最高抗菌活性达27.9 mm。与初始的发酵条件相比,人LL-37基因与IFN-α2a融合蛋白的产量提高了32.29%。     

不同控制模式对毕赤酵母表达IL-2-HSA的比较研究

以溶氧（DO）和甲醇浓度作为控制指标,分别考察了诱导表达阶段DO和甲醇浓度对毕赤酵母高密度发酵表达白介素-2-白蛋白融合蛋白（IL-2-HSA）的影响。通过实验比较得出：诱导阶段控制DO对IL-2-HSA表达影响不明显,而甲醇浓度对IL-2-HSA影响很大。高甲醇浓度（15～25g／L）有利于IL-2-HSA的表达,发酵结束时的表达量约是低甲醇浓度（5～8g／L）诱导下的2倍。此外,在诱导阶段,通过限制性添加甘油可以有效提高菌体的呼吸活性,促进菌体生长。     

重组人白细胞介素-1α在毕赤酵母中的表达、纯化及生物学活性检测

在毕赤酵母中分泌表达重组人白细胞介素-1α(rh IL-1α),优化rh IL-1α的发酵工艺及纯化方法,以获得高表达、高纯度具有生物学活性的rh IL-1α。通过PCR扩增获得h IL-1α基因,构建其真核表达载体p PICZαA/h IL-1α,电转化至毕赤酵母X-33,用PCR和SDS-PAGE方法筛选高效表达rh IL-1α的工程菌株并进行Western blot鉴定,DEAE弱阴离子离子交换层析一步纯化表达产物,并用MTT法初步检测其对人肝癌细胞7402的生物学作用。rh IL-1α在摇瓶规模下,经甲醇诱导4 d后表达量约为30 mg/L。Western blot检测rh IL-1α的特异性结合,获得纯度约95%,收率40%左右的rh IL-1α,并证明rh IL-1α能够抑制人肝癌细胞7402的增殖。构建了重组h IL-1α的基因工程菌,并在毕赤酵母中实现了高效表达,为进一步研究其生物学活性和功能奠定了基础。     

人源抗狂犬病毒糖蛋白稳定型抗体精氨酸密码子突变株的构建及其在巴斯德毕赤酵母中的分泌表达

目的：对人源抗狂犬病毒糖蛋白（GPRV）单链二硫键稳定抗体（ScdsFv）进行精氨酸密码子修饰,实现其在酵母中的分泌表达,并检测其生物学活性。方法：参照巴斯德毕赤酵母偏好密码子,对抗GPRV ScdsFv原核表达基因进行密码子修饰,并通过点基因融合技术构建ScdsFv重组酵母表达基因,连接pPIC9K构建重组表达质粒pPIC9K-ScdsFv,电转化毕赤酵母GS115,经筛选后进行甲醇诱导表达。结果：SDS-PAGE及Western blot检测到重组表达质粒在30℃经甲醇诱导表达的蛋白相对分子质量为30000;荧光抗体试验验证ScdsFv能靶向结合GPRV;MTT试验说明ScdsFv能中和狂犬病毒,具有一定的细胞保护作用。结论：重组ScdsFv在酵母系统中可以有效表达。具有较好的生物学活性。     

人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

利用毕赤酵母(Pichia pastoris)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein6,h Lyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成h Lyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的p PIC9K质粒构建重组表达载体p PIC9K-hlyzl6;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDSPAGE检测在14.8 k Da处有重组h Lyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定h Lyzl6酶学活性,结果表明h Lyzl6对溶壁微球菌(Micrococcus lysodeikticus)有较好的杀灭作用,最适反应温度为40℃,最适p H为5.5,其酶活力为54 700U/mg,Cu2+对其活性有明显抑制,EC50为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组h Lyzl6,证实其在体外具有杀菌活性,初步揭示h Lyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究h Lyzl6的功能和应用开发奠定了基础。     

利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2

利用甘油醛三磷酸脱氢酶(glyceraldehydes-3-phosphatedehydrogenase,GAP)启动子在毕赤酵母中表达人鹅型溶菌酶2(human goose-type lysozyme 2,h LysG2),并在小试规模建立一套有效的重组hLysG2(recombinant h LysG2,rh LysG2)生产工艺流程。根据毕赤酵母密码子偏爱性设计并人工合成hLysG2基因,将其连接至pGAPZαA质粒中,构建重组表达质粒pGAPZαA-h LysG2。将重组表达载体线性化后电转化毕赤酵母GS115感受态细胞,通过Zeocin抗性筛选获取高拷贝重组菌株,并在5L生物反应器中进行发酵培养。发酵60h后发酵液上清酶活性达到最高,发酵液上清经SDS-PAGE及Western blot检测证实rh LysG2得到表达。与诱导型表达相比,组成型表达发酵时间缩短了48h,上清中rhLysG2总活性提高了23.8%;使用甲壳素亲和层析和分子筛层析对rhLysG2进行纯化后,每升发酵液上清可纯化到187.4mg重组蛋白,纯化产物纯度达99.0%以上;浊度测定法分析显示,在p H 5.6、30℃和0.1mol/L Na+的条件下,rhLysG2可达到最大酶活性13 500U/mg。利用GAP启动子在毕赤酵母中成功表达了高纯度和高活性的rh LysG2,避免了甲醇的使用,缩短了发酵时间,提高了蛋白产量,为将rhLysG2开发为新型抗耐药菌药物奠定了基础。     

乙型肝炎病毒PreS2S基因在毕赤酵母中表达的研究

构建重组质粒PHIL-D2/PreS2S以研究乙肝病毒PreS2(120-146)S基因编码蛋白在毕赤酵母中的表达。通过PCR扩增获得PreS2S片段,插入含AOX1启动子的Pichia Pastoris表达载体PHIL-D2中,构建重组表达质粒PHIL-D2/PreS2S,转化酵母宿主菌GS115。挑取阳性克隆摇床培养,甲醇诱导表达。通过ELISA、RPHA鉴定表达产物。成功构建了PHIL-D2/PreS2S真核表达载体,经过序列分析,插入的基因为在中国流行的adr亚型。在毕赤酵母中重组载体表达了S蛋白,S蛋白的表达量为34.9 mg/L,PreS2抗原检测为强阳性。利用毕赤酵母表达系统能够有效地表达乙型肝炎病毒的PreS2S蛋白,PreS2S蛋白具有良好的生物学活性。     

Cytochalasin-B-induced immunosuppression of murine allogeneic anti-tumor response and the effect of recombinant human interleukin-2

Summary Cytochalasin B (CB), administered i.p. to C57B1/6 mice in a single dose as a suspension in carboxymethylcellulose 2%/Tween 20 1%, inhibits in a dose-dependent and time-dependent manner the ability of spleen cells to respond to allogeneic P815 mastocytoma tumor cells in vitro. Spleen cells from CB-treated animals sensitized to X-irradiated P815 cells in 4-day cultures at a 50 : 1 responder: stimulator ratio and tested for specific cytotoxicity against⁵¹Cr-labelled P815 target cells showed strong inhibition 3 h after CB treatment at a dose of 50 mg/kg. A dose of 25 mg/kg showed measureable but not statistically significant inhibition at 3 h, whereas 10 mg/kg produced only slight inhibition, and 5 mg/kg and 2 mg/kg were non-inhibitory. None of the doses produced significant suppression 19 h or 72 h after CB treatment. Addition to the sensitization cultures of human recombinant interleukin-2 (rhIL-2) at 350 BRMP units/ml completely restored tumor lytic capacity. C57B1/1 mice treated with CB 50 mg/kg, i.p. and challenged i.p. with 3 × 10⁷ allogeneic P815 mastocytoma cells showed a brief, time-dependent, statistically significant abrogation of allogeneic responsiveness consistent with transient reversible immunosuppression within 3–12 h following CB treatment. No such inhibition of host allogeneic responsiveness in vivo was observed when CB was administered 24 h prior to, simultaneously with, or 1, 2, or 4 days after tumor challenge. Thus CB at the highest tolerated i. p. dose in vivo causes only a transient inhibition of anti-allo-responsiveness measured in culture, and rhIL-2 used in vitro restores lytic capacity. The anti-allo effect of CB is also seen to be transient directly in vivo since allogeneic tumor outgrowth is permitted for only a brief period following administration of CB. These results indicate that the use of CB in vivo in anti-tumor chemotherapy protocols will not be complicated by profound or prolonged immunosuppressive effects.Supported by the Stella Hardeman Memorial Grant for Cancer Research to the American Cancer Society and by the Liposome Company, Princeton, New Jersey, USA     

Prediction on the binding domain between human interleukin-6 and its receptor

Based on the spatial conformations of human interleukin-6 (hIL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hIL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hIL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hIL-6 and hIL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hIL-6 and hIL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hIL-6 and designing novel antagonist with computer-guided techniques.     

Prediction on the binding domain between human interleukin-6 and its receptor

Based on the spatial conformations of human interleukin-6 (hlL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hlL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hlL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hlL-6 and hlL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hlL-6 and hlL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hlL-6 and designing novel antagonist with computer-guided techniques.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

Simple purification ofEscherichia coli-derived recombinant human interleukin-2 expressed with N-terminus fusion of glucagon

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed inEscherichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced inE. coli cytoplasm were easily dissolved by simple alkaline pH shift (8→12→8). Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.     

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method. This revised version was published online in August 2006 with corrections to the Cover Date.     

小鼠皮肤损伤修复过程中白细胞介素-6对某些炎症因子基因表达的影响

为了解在皮肤损伤修复过程中白细胞介素-6(Interlukin-6,IL-6)对其他炎症因子基因表达的影响,以及对皮肤损伤修复过程的影响,用免疫组织化学染色法和RT—PCR法,检查了IL-6基因敲除鼠(IL-6^-/-鼠)和正常野生型鼠(IL-6^ / 鼠)皮肤损伤后损伤区组织内不同时间的白细胞介素-1α(Interlukin-1α,IL-1α)、白细胞介素-1β(Interlukin-1β,IL-1β)、角质细胞诱导因子(Keratinocyte chemoattractant,KC)、单核细胞诱导蛋白-1α(Macrophage inflammatory protein-1α,MIP-1α)以及单核细胞诱导蛋白-2(Macrophage inflammatory protein-2,MIP-2)这5种炎症因子的基因表达的变化。结果发现：不论是IL-6^-/-鼠还是IL-6^ / 鼠,其被检因子的基因表达都以第3d为高峰,第6d则明显下降;同时,在损伤后的第3d和第6d,IL-6^-/-鼠的5种因子的基因表达水平显著低于IL-6^ / 鼠,而在损伤后的第1d,只有MIP-1α和KC的水平低于IL-6^ / 鼠,而且,IL-6^-/-鼠的皮肤损伤修复过程也稍迟于IL-6^ / 鼠,但皮肤的损伤仍可完全修复。上述结果显示,IL-6在小鼠皮肤损伤过程中诱导其他5种被检因子的产生,从而促进皮肤损伤的修复,但IL-6基因缺失也不会严重影响皮肤损伤的修复。     

Interleukin-1β induction of tissue inhibitor of metalloproteinase (TIMP-1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts

Elevated levels of tissue inhibitor of metalloproteases-1 (TIMP-1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂), in the control of TIMP-1 synthesis and expression in human synovial fibroblasts. Recombinant human IL-1β induced a time- and dose-dependent saturable response in terms of TIMP-1 mRNA expression (effective concentration for 50% maximal response, EC₅₀ = 31.5 ± 3.3 pg/ml) and protein synthesis (EC₅₀ = 30 ± 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H-7, staurosporine, and calphostin C, reversed the rhIL-1β induction of TIMP-1 mRNA. PGE₂ also inhibited rhIL-1β-stimulated TIMP-1 mRNA expression and protein secretion in a dose-dependent fashion. The concentration of PGE₂ necessary to block 50% of rhIL-1β-stimulated TIMP-1 secretion, IC₅₀, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE₂. The phorbol ester, PMA, up-regulated considerably the mRNA expression of TIMP-1 but had no effect on protein production. Calphostin C substantially reduced PMA-activated TIMP-1 expression. Staurosporine, calphostin C, H-7, and substances that elevate cellular levels of cAMP, like PGE₂, also reduced basal expression and synthesis of TIMP-1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP-1 expression and secretion in human synovial fibroblasts.     

Different individual immune responses elicited by in vitro immunization

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.