人白细胞介素-11基因在家蚕培养细胞和虫体内的表达

将缺少编码信号肽序列的人白细胞介素－11（hIL-11)546核苷酸cDNA,重组于质粒pBacPAK8构建重组转移载体pBacIL-11,与经线性化修饰的家蚕核型多角体病毒（BmBacPAK)DNA共转染家蚕培养细胞株BmN,获得了插入hIL-11基因的重组病毒。Southern杂交表明重组病毒基因组中含有hIL-11基因片段,RNA斑点杂交表明hIL-11基因得到了转录。重组病毒感BmN细胞株、家蚕幼虫和蛹,在细胞培养上清、细胞抽提物、幼虫和蛹的体液样品中,SDS－PAGE电泳分析都能检测得到表达产物的特异性条带;采用IL－11依赖细胞株B9－11和MTT法测定表达产物的生物活性,表明rIL-11基因分别在培养细胞和蚕体内得到了高效表达。     

人白细胞介素11 cDNA的克隆、融合表达及活性测定

取人胎肺做原代培养细胞,ＰＭＡ诱导后,利用ＲＴ－ＰＣＲ技术,扩增出去了Ｎ端信号肽序列的ＩＬ１１ｃＤＮＡ,经序列分析表明,该序列与文献报道序列高度同源,仅３个核苷酸有变化,氨基酸序列一致。将此ＩＬ－１１ｃＤＮＡ克隆人硫氧还蛋白基因融合表达载体ｐＴＲＸＦＵＳ的ｔｒｘＡ基因３‘末端,利用其３’端的蛋白肠激酶切点。构建符合读码框的融合基因。该融合蛋白在大肠杆菌中表达量法２０％以上。用ＩＬ－６依赖细胞株７Ｔ     

人白细胞介素11的定点聚乙二醇修饰

利用人白细胞介素11（hIL-11）无半胱氨酸（Cys）残基这一特点,通过定点突变将一个Cys残基引入hIL-11的N末端。然后,利用与Cys 巯基特异性反应的mPEG-马来酰亚胺将mPEG偶联到预先选定的位点,经层析纯化得到hIL-11的定点PEG修饰物。利用依赖型细胞株7TD1测定其生物学活性,结果表明,其体外生物学活性保持原有hIL-11活性的30%左右。定点聚乙二醇修饰方法为定向改造hIL-11,提高其药效的应用研究打下基础。     

Production and purification of recombinant human interleukin-5 from yeast and baculovirus expression systems

A cDNA for human interleukin-5 (hIL-5) was created from the hIL-5 gene using site-directed mutagenesis to splice out the introns in vitro. This cDNA was expressed in yeast and baculovirus systems, utilizing in both cases an in-frame fusion to the pre sequence of the alpha-mating-type factor to direct secretion. The highest level of production was achieved from Sf9 cells using a baculovirus vector in serum-containing medium (2.7 mg/l), whereas in serum-free medium ten times less hIL-5 was produced. In the yeast system much lower levels of hIL-5 were produced (12.5 micrograms/l). Recombinant hIL-5 was purified to homogeneity from serum-free baculovirus cultures. The rhIL-5 consisted of a 30-kDa homodimer linked by disulfide bridging. The purified recombinant protein had a specific activity on murine BCL1 cells of 1.5 x 10(4) U/mg, of 3 x 10(5) U/mg in the murine eosinophil differentiation factor assay, and 2.4 x 10(7) U/mg in a human peripheral eosinophil maintenance assay.     

SUBCUTANEOUS OR ORAL ADMINISTRATION OF rhIL-11 MODULATES THE CONTACT HYPERSENSITIVITY RESPONSE

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.     

Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli

Previously, we constructed human interleukin-6 (hIL-6)-secreting Escherichia coli and Salmonella typhimurium strains by fusion of the hIL-6 cDNA to the HlyA(s) secretional signal, utilizing the hemolysin export apparatus for extracellular delivery of a bioactive hIL-6-hemolysin (hIL-6-HlyA(s)) fusion protein. Molecular analysis of the secretion process revealed that low secretion levels were due to inefficient gene expression. To adapt the codon usage in hIL-6 cDNA to the E. coli codon bias, a synthetic hIL-6Ec gene variant was constructed from 20 overlapping oligonucleotides, yielding a 561-bp fragment, which comprises the complete hIL-6 cDNA sequence. Genetic fusion of the hIL-6Ec gene with the hlyA(s) secretional signal as an integral part of the hemolysin operon resulted in 3-fold higher hIL-6-HlyA(s) secretion levels in E. coli, compared to a strain expressing the original hIL-6-hlyA(s) fusion gene. An increase in the electrophoretic mobility of secreted hIL-6-HlyA(s) in non-reducing SDS-PAGE, similar to that found for recombinant mature hIL-6, and the absence of such a mobility shift in the intracellular hIL-6-HlyA(s) protein fraction indicated that in hIL-6-HlyA(s) most probably correct intramolecular disulfide bond formation occurred during the secretion step. To confirm the disulfide bond formation, hIL-6-HlyA(s) was purified by a single-step immunoaffinity chromatography from culture supernatant in yields of 18 microg/L culture supernatant with purity in the range of 60%. These results demonstrate that codon usage has an impact on the hemolysin-mediated secretion of hIL-6 and, furthermore, provide evidence that the hemolysin system enables secretory delivery of disulfide-bridged proteins.     

人IL-29的定点突变及抗肿瘤活性初步分析

为探讨人白细胞介素-29(h IL-29)变异体的抗肿瘤活性,根据h IL-29成熟肽的生物信息学分析数据,采用大引物PCR方法对其肽链第33位赖氨酸、35位精氨酸的编码基因进行定点突变,获得的h IL-29变异体基因构建重组真核表达质粒转化毕赤酵母(Pichia pastoris)GS115进行发酵表达,经纯化得到重组人白细胞介素-29变异体蛋白(rh IL-29mut33,35)。经CCK-8法检测抗肿瘤细胞增殖的数据显示,rh IL-29mut33,35对肝癌细胞BEL7402、结肠癌细胞HCT8和胃癌细胞SGC7901的增殖均具有抑制作用,高剂量组对这3种肿瘤细胞的增殖抑制率分别为(30.99±1.58)%、(22.47±1.37)%和(32.05±2.02)%,而且抗增殖作用比野生型rh IL-29的更强(P0.01),表明变异体rh IL-29mut33,35具有潜在的医药开发价值。     

应用痘苗病毒为载体表达人白细胞介素4的研究

在真核细胞中表达近似于人类天然糖基化的白细胞介素４（ｈＩＬ－４）,为重要的研究课题。利用基因工程技术,以痘苗病毒作为载体,使之在真核细胞中表达ｈＩＬ－４,为生产糖基化的ｈＩＬ－４开辟新的可能途径。在痘苗病毒表达载体ｐＪ１２０质粒的基础上,组建了含有ｈＩＬ－４基因的ｐＪｌ２０／ｈＩＬ－４重组质粒,该质粒以痘苗病毒的胸腺嘧啶核苷激酶（ＴＫ）基因为侧翼序列,中间插入痘苗病毒的Ｐ１１和Ｐ２５两个转录方向相反的启动子,Ｐ２５的下游连接β－半乳糖苷酶（ＬａｃＺ）基因,ｈＩＬ－４基因在痘苗病毒启动子Ｐ１１控制下进行表达,将ＰＪ１２０／ｈＩＬ－４质粒ＤＮＡ用脂质体转染法导入ＴＫ阴性的骨髓瘤细胞（ＴＫ１４３细胞）,与预先感染的痘苗病毒在细胞内进行同源序列重组,并且用病毒的核酸杂交鉴定证实,ｈＩＬ－４基因已重组到痘苗病毒基因组中,用ｈＩＬ－４应答细胞株检测病毒感染细胞上清,发现重组病毒感染的细胞上清中含有高滴度的ｈＩＬ－４活性,并对此种情况下ｈＩＬ－４产生的条件进行了动态观察。     

Non-core region modulates interleukin-11 signaling activity: generation of agonist and antagonist variants

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.     

Interleukin-11 promotes the progress of gastric carcinoma via abnormally expressed versican

Versican, a ubiquitous component of the extracellular matrix (ECM), accumulates both in tumor stroma and cancer cells and is highly regulated by various cytokines. The aberrant expression of versican and its isoforms is known to modulate cell proliferation, differentiation, and migration, all of which are features of the invasion and metastasis of cancer; versican is also known to favour the homeostasis of the ECM. Interleukin-11 (IL-11) is an important cytokine that exhibits a wide variety of biological effects in gastric cancer development. Here, we analysed the expression of versican isoforms and found that the major isoforms expressed by both gastric carcinoma tissue and gastric cell lines were V0 and V1, and V1 was significantly higher in gastric carcinoma tissue. The treatment of the gastric cell lines AGS and MKN45 with rhIL-11 resulted in a significant increase in the expression of V0 and V1. Exogenous IL-11 increased migration in AGS and MKN45 cells, whereas these effects were reversed when the expression of V0 and V1 were abolished by siRNA targeting versican V0/V1. Collectively, these findings suggest that the abnormally expressed versican and its isoforms participate, at least in part, in the progress of gastric carcinoma triggered by IL-11.     

毕赤酵母表达重组人白细胞介素11的连续培养研究

对毕赤酵母表达重组人白细胞介素11(rhIL11)工程菌的连续培养进行了工艺研究。连续培养在10L工作体积的发酵罐中进行,整个发酵过程历时约20天,发酵上清中rhIL11表达浓度约400mgml。在稀释率D=005h下,菌浓OD600达到100以上。     

Mechanism of the antiulcerogenic effect of IL-11 on acetic acid-induced gastric ulcer in rats

The purpose of this study was to evaluate the healing effect of interleukin-11 (IL-11) on acetic acid-induced gastric ulcer in rats. Gastric ulcers were induced in male Wistar rats by applying acetic acid to the fundus of the stomach. Recombinant human interleukin-11 (rhIL-11 100 microg/kg/twice daily, subcutaneously) was administered starting on the 2nd day before ulcer induction up through the 7th day after ulcer induction. Control rats were injected with bovine serum albumin. At 12 hours and 7 days after ulcer induction, the animals were sacrificed, and the ulcer index, proliferating cell nuclear antigen (PCNA) expression, and IL-11alpha receptor expression in the gastric tissues were studied. The ulcer index of the rhIL-11-treated rats was significantly lower than that of the control rats at the 7th day. The expression of PCNA as evaluated by Western blotting and immunohistochemistry, was enhanced in both the mucosal proliferative zone and proper muscle layer of the rhIL-11-treated rats in comparison with that in the control rats. IL-11alpha receptor expression was observed in the mucosal neck cells of the rhIL-11-treated rats and control rats. These findings suggest that IL-11 accelerates ulcer healing by inducing the proliferation of mucosal and muscular cells.     

Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris

Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.     

水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的, OsNPSN11是从水稻中克隆的Qb-SNARE家族基因, 文章将OsNPSN11构建到原核表达载体pET-30a中与6个His标签融合, 重组质粒pET-OsNPSN11转化BL21(DE3)0.5 mmol/L IPTG诱导4 h后获得了高效表达。用镍离子亲和树脂(Ni²⁺-NTA His Bind Resin) 纯化融合蛋白, 以纯化后的蛋白为抗原免疫新西兰家兔制备多克隆抗体, Western blotting结果显示, 该抗体能特异识别在原核系统表达的抗原, 以及水稻不同组织质膜组分中的OsNPSN11, 可用于转基因水稻中目标蛋白的表达分析。     

Engineering and use of (32)P-labeled human recombinant interleukin-11 for receptor binding studies.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.     

Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (C_max) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.     

人白细胞介素22基因的分离及其在大肠杆菌中的克隆与表达

白细胞介素 2 2 (ＩＬ 2 2 )是 2 0 0 0年发现的一种新的人类细胞因子 ,它在多种组织中均有表达 ,包括胰腺、脑 ,肝、小肠、直肠和肾等 .在功能方面 ,ＩＬ 2 2上调肝细胞急性期产物 ,参与炎症反应[1～ 3] ;诱导胰腺相关蛋白ＰＡＰ1 Ｒｅｇ2和ＯＰＮ的高表达 ,表明ＩＬ 2 2参与胰腺免疫功能反应[4 ] ;它对ＣｏｎＡ刺激下Ｔｈ1细胞ＩＦＮ γ的产生没有明显的抑制作用 ,却大大抑制了Ｔｈ2细胞ＩＬ 4的分泌 ,这对于哮喘有潜在的治疗作用[1] .我们利用反转录ＰＣＲ获得了编码ｈＩＬ 2 2成熟蛋白的ｃＤＮＡ ,并构建了高表达载体ｐＢＶｈＩＬ 2 2 ,…     

Subcutaneous or oral administration of rhIL-11 modulated the contact hypersensitivity response

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.