【作者】 孔宁；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 抑瘤素M(oncostatin M)作为一种多功能的细胞调节因子,可作用于多种细胞,具有调节基因活性、细胞存活、增殖和分化的潜在功能,在炎症反应、造血作用、肝脏发育以及免疫系统等方面展示出独特的生物学活性,具有重要的生物学作用。本研究利用基因拼接方法,以人胚胎组织DNA为模板,通过PCR技术获得了成熟hOSM基因序列,构建了成熟hOSM的毕赤酵母真核表达载体pPICZαC-hOSM。电转化毕赤酵母X-33后提取酵母基因组DNA,使用特异性PCR引物进行PCR鉴定,证实成熟hOSM基因已经稳定整合到酵母基因组中,从而建立了hOSM毕赤酵母的高效表达体系。取其培养液上清经SDS-PAGE和Westernblot证实获得了相对分子量约为30kDa的rhOSM蛋白。通过SDS-PAGE和Westernblot方法筛选出能够稳定、高效分泌表达rhOSM的酵母工程菌。并首次利用80L发酵罐进行rhOSM的大规模发酵,摸索出其最佳发酵条件,即在发酵液pH5.4的条件下,甲醇诱导表达36h时放罐,rhOSM产量可达100mg·L-1,国内外未见相关报道。并采用SP Sepharose XL阳离子交换树脂及SourceTM30RPC反相疏水柱层析对其发酵产物进行纯化,纯度可达95%,收率可达50%。本实验为下一步进行工业化生产及进一步探究OSM在体内体外的生物学作用及其潜在的临床应用奠定基础。更多还原

【Abstract】 In the last two decades, a large number of cytokines have been found and areclassified into several families based on their structural properties as well as theirreceptor components. Oncostatin M (OSM), a glycoprotein monomer of 28,000Da,was originally isolated and purified from the conditioned media of phorbol12-myristate 13-acetate (PMA)-stimulated human histiocytic lymphoma U937cells by Zarling et al and was named by its activity to inhibit the proliferation ofA375 melanoma cells. At first, all people knew about OSM was its abilities ofinhibiting some human tumor cells, inducing other tumor cells differentiation andstimulating the growth of normal human fibroblasts. However, with the progressof studying of OSM, people find that OSM is a multifunctional cellular regulatorand can act on a wide variety of cells, which has potential roles in the regulationof gene activation, cell survival, proliferation and differentiation. Furthermore,accumulating evidence now indicates that OSM exhibits many unique biologicalactivities in inflammation, hematopoiesis, liver development and immune system.OSM belongs to the Interleukin(IL)-6 subfamily of cytokines, whose membersinclude LIF, G-CSF, CNTF, IL-6, IL-11, CT-1, et al. The main property of thefamily is its function redundancy. Among the family members, OSM is mostclosely related to LIF structurally, functionally and genetically.All members of IL-6 subfamily share overlapping biological functions, thestructural foundation of function redundancy is based on their shared receptorsubunit, gp130. There are two types of OSM receptor, the typeⅠand the typeⅡ.The typeⅠOSMR is identical to LIF receptor(LIFR) and the typeⅡOSMR isspecific to OSM. OSM displays some specific biological properties throngh thetypeⅡOSMR. After binding OSMR, OSM activates Jak/STAT and Ras/Raf/MAPK singal transducer cascades, therefore, OSM regulates related genetranscription.So far, most of the present reports about OSM are the new roles of OSM inhuman body. All in all, OSM plays a very important role in many reactions ofhuman body as a pleiotropic cytokine. Among numerous biological functions ofOSM, many have the clinical therapeutic value such as inhibiting the proliferationof many human tumor cells;OSM gene transferring or inject OSM proteindirectly can induce nude mouse to create T lymph cell in peripheral lymph follicle,which can recreate T lymph cell immune deficiency;OSM can act as aanti-inflammatory factor to regulate inflammation response.At present, all we can buy recombinant human OSM (rhOSM) and polyclonalantibody of OSM in the market are expressed in E.coli. Additionally, human OSMalso can expressed in mammal cell eukaryotic expression system but two methodshave some insurmountable flaws that limit the large-scale manufacture. So wedecide to select Pichia pastoris that is suitable for large-scale fermentation forindustrial manufacture to build up rhOSM eukaryotic expression system toproduce rhOSM.1. Clone and identification of the hOSM geneAccording to hOSM gene structure and the mature OSM inhibitive ability is5～60 times of precursor OSM, we used the human embryonic DNA as templet toobtain a sequence including the second exon , the second intron and the third exonvia PCR. And we used this sequence as templet to obtain the mature hOSM genesequence from the second exon and the third exon respectively via PCR. Then, theexpression vector of Pichia pastoris pPICZαC-hOSM was constructed by genejointing. The aforementioned plasmid was authenticated by endonucleasedigestion and sequencing. In order to introduce the endonuclease site to joint thetwo exons, we modified the four individual bases which don’t change the aminoacid sequence, the changed bases are as follows: 233c→a,235t→a,238c→t,229t→c. And the other sequence was identical with the sequence of hOSM cDNAin GeneBank.2. Construction of Pichia pastoris high efficient expression system for rhOSM(1) Screening of the transformed Pichia pastoris strainsLinearize the expression vector pPICZαC-hOSM after confirmation byendonuclease digestation assay and sequencing and transform it into Pichiapastoris X-33 via electroporation. Extract the genomic DNA of the transformedyeasts to perform PCR using the specific expression primers. The results showthat the resulting DNA was only showed in the transformed yeasts, and there isnot the predicted DNA in the control groups including yeasts without thetransformed hOSM gene and yeasts transformed blank plasmid. This indicatedthat hOSM gene was stably transformed into Pichia pastoris genome DNA.(2) SDS-PAGE of the hOSM proteinProliferate the PCR tested positively yeast clones, then induced the expressionof rhOSM with methanol. The supernatant of before inducement and afterinducement were analyzed by SDS-PAGE. The resulting protein bands of 30kDawas only present in the supernatant of inducement with methanol and there is notthe predicted protein bands in the control groups. The amount reaches to the peakduring 72～120 hours of inducement while decreasing after 120 hours. And therewere different expression quantities among the different clones.(3) Western blot of the hOSM proteinThe supernatant of before inducement and after inducement were analyzed byWestern blot. The resulting protein bands was only present in the supernatant ofinducement with methanol and there is not the predicted protein bands in thecontrol groups. And there were different expression quantities among the differentclones. Maybe the extraneous gene integrated into the different sites of the yeast’schromosome, and the transcript efficiencies were different.3. Studies on large-scale fermentation and purification process of rhOSM(1)Studies on large-scale fermentation process of rhOSMCompared with E.coli and mammal cell eukaryotic expression system, Pichiapastoris has many advantages as a kind of expression host, and it’s very suitablefor large-scale expression of the extraneous proteins. So we first explored thelarge-scale fermentation process of rhOSM and found that the suitable pH ispH5.4, DO above 25% and the supply speed of methanol is 9.3ml/h/L initialfermentation volume. The concentration of rhOSM in the broth can reached100mg·L-1.(2) A new method to purify rhOSMCentrifugate the fermentation product and collect the supernatant. Then thesupernatant was purified by SP Sepharose XL on pH4.0 and SourceTM30RPC andthen collect the eluate. The degree of purity could be more than 95% and the yieldcoefficient was higher than 50%.From the above mentioned,the eukaryotic expression plasmid pPICZαC-hOSM and Pichia pastoris high efficient expression system for rhOSM wereconstructed successfully and we screened the high efficient yeasts strains.Therefore, we obtain rhOSM which is identical to the native OSM. We firstexplored large-scale fermentation process of rhOSM in 80 liter fermentator and anew purification process, which piered foundation to carry out industrialmanufacture of OSM and facilitated further studies on the other properties,biological activities of OSM in vivo and in vitro and potential clinical therapeuticapplications of OSM.更多还原