201247874 六、發明說明： 【發明所屬之技術領域】 本發明係有關於一種骨形成蛋白、2(b(^ morphogenetic protein-2 ; BMP-2)的製備方法，特別有關於 藉由改變培養基成分來增加骨形成蛋白-2的產量。 ' 【先前技術】 骨形成蛋白’簡稱為BMPs ’為骨基質中的醣蛋白〒胜 肽，其包含雙硫鍵結構。骨形成蛋白的分子旦： J刀丁里介於 26,000-32,000 ’其為一生長因子家族，彼此具有類似的择 構及功能。至今，已發現43種骨形成蛋白。骨形成蛋白為 一種區域性的生長因子，可單獨促進骨組織的形成，促使 未分化間葉細胞(mesenchymal cells)進入軟骨及骨。骨开^成 蛋白具有各種促進骨生成的能力。 BMPs的能力不限於骨生成，在其他組織，例如腦及腎 臟也可發現高濃度的BMPs。已有相關文獻揭露BMPs在發 生及分化上可能也扮演重要的角色（Wal.1，N. A., Blessing, M., Wright, C. V. E., and Hogan, B. L. M., J Cell Biol., 120： 493-502 (1993); Ozkaynak, E., Schnegelsberg, P. N. J., Jin, D. F·，Clifford, G. M·, Warren，F. D.，Drier，E. A.，and Oppermann, H., J. Biol. Chem., 267: 25220-25227 (1992); Lyons, K. M.，Jones，C. M·，and Hogan, B. L· M., Trends in Genetics, 7: 408-412 (1991))。目前，BMPs 已被證實可促進 神經細胞分化（Basler, K., Edlund，Τ·, Jessell，Τ. Μ·, and Yamada, Τ., Cell, 73: 687-702 (1993); Paralkar, V. Μ., 201247874201247874 VI. Description of the Invention: [Technical Field] The present invention relates to a method for preparing bone morphogenetic protein 2, b (^ morphogenetic protein-2; BMP-2), in particular, by changing the composition of the medium Increase the production of bone morphogenetic protein-2. '[Previous technique] Bone morphogenetic protein 'abbreviated as BMPs' is a glycoprotein acetopeptide in the bone matrix, which contains a disulfide bond structure. Molecular dentin of bone morphogenetic protein: J knives It is between 26,000 and 32,000 ', which is a family of growth factors with similar selection and function. Up to now, 43 bone-forming proteins have been discovered. Bone morphogenetic protein is a regional growth factor that can promote bone tissue alone. It forms and promotes undifferentiated mesenchymal cells into cartilage and bone. Osteoproteoprotein has various abilities to promote bone formation. The ability of BMPs is not limited to bone formation, but can also be found in other tissues such as brain and kidney. Concentrations of BMPs. It has been revealed in the literature that BMPs may also play an important role in the occurrence and differentiation (Wal.1, NA, Blessing, M., Wright, CVE, and Hog). An, BLM, J Cell Biol., 120: 493-502 (1993); Ozkaynak, E., Schnegelsberg, PNJ, Jin, D. F., Clifford, G. M., Warren, FD, Drier, EA, and Oppermann, H., J. Biol. Chem., 267: 25220-25227 (1992); Lyons, KM, Jones, C. M., and Hogan, B. L. M., Trends in Genetics, 7: 408- 412 (1991)). Currently, BMPs have been shown to promote neuronal differentiation (Basler, K., Edlund, J., Jessell, Τ. Μ·, and Yamada, Τ., Cell, 73: 687-702 (1993) ); Paralkar, V. Μ., 201247874
Weeks，B. S·, Yu，Y. M.，Kleinman，Η· K.，and Reddi, A. H.， J. Cell Biol·，119: 1721-1728 (1992))。 在整個BMP家族中，以BMP-2具有廣泛的骨生成能 力，且被研究的最為透徹。具有骨生成活性的重組人類 BMP-2已被證明具有安全性，可應用於骨的治療及再生。 BMP-2的應用對傳統骨科、整型外科、牙周和顱面重 建手術產生重大的影響。目前獲得BMP-2的方法主要有二 種，分別為由脫弼皮質骨（demineralized cortical bone)萃取 BMP-2蛋白，以及利用表現系統表現重組BMP-2蛋白。 由骨組織分離BMP-2蛋白之方法需要大量的骨組織且 產量非常低，約每40 kg的牛骨粉僅可獲得40 pg的BMP 混合物（Wozney et al.，Science 242 (1988), 1528-1534)。再 者’若由人骨組織分離BMP-2蛋白，會產生道德上的問題， 且會有致病源汙染的風險（賈庫氏症、HCV等）。同樣地， 由豬或牛分離BMP同源蛋白也會產生類似的汙染風險。 因此，目前主要是利用表現重組BMP-2來獲得BMP-2 蛋白’表現方法包括真核表現系統及原核表現系統。 原核表現糸統的優點為操作簡單、成本低，且可獲得 較高的產量。相對於真核表現系統，原核細胞無法表現正 確的BMP-2前驅物，因此難以形成成熟的BMP-2蛋白。 真核表現糸統的缺點為需要較南的技術且產量較低。 雖然真核表現系統可產生具活性的BMP-2蛋白，但真 核表現系統的產量極低。先前有文獻指出添加低分子量 (5,000 1^>¥)及高分子量（500,000]\^)的硫酸葡聚醣可增加 BMP-2蛋白的產量，但其產量仍顯不足。因此業界亟需一 s 4 201247874 種可增加bmp產量的方法。 【發明内容】 本發明係提供一種骨形成蛋白_ 供-細胞株H 4方法，包括知 培養於-培養基令，41=:基因’將此細跑株 括提=r 一: 株培養於一培:基;1;==:基因’將該細胞 /、f此°養基包括硫酸葡聚醣。 明顯明之上述和其他目的、特徵、和優點能更 細，並配合所附圖示，作詳 【實施方式】 本發明係提供―種骨形成蛋白_2的製備方法，包括提 一、、’用胞株’其含有骨形成蛋白_2的基因，將此細胞株於 ，田的條件下培養於—培養基中，其中此培養基包含硫 I匍聚醣。 ^本發明所述之‘‘細胞株”係指任何用於蛋白質表現的 細胞株，包括動物細胞、低等真核生物或原核生物，較佳 為動物細胞。動物細胞包括，但不限於，猴COS細胞、CHO 、田胞、人類腎臟293細胞、人類表皮A431細胞、人類 C〇l〇205細胞、3T3細胞、CV-1細胞、其他經轉殖的靈長 類細胞、正常的雙套細胞、由體外培養之原生組織衍生細 胞株、原生移殖體、HeLa細胞、老鼠L細胞、BHK、HL-60、 201247874 U937、HaK、PerC6、NOS 或 Jurkat 細胞等。在一實施例 中’本發明之細胞株較佳為CHO細胞。 此細胞株中具有骨形成蛋白-2(以下簡稱BMP-2)基 因，此BMP-2基因可為此細胞自源性的BMP-2基因，也 可為轉染至細胞内的外源性BMP-2基因或重組BMP-2基 因。BMP-2基因可來自人類或非人類動物（例如’小鼠、大 鼠、倉鼠、兔子或駱駝等）。在一實施例中，此BMP_2基 因為人類BMP-2基因，並將此bmp-2基因轉染至CHO細 胞中。 本發明中所述之“適當條件”係指可促進細胞存活、 細胞生長及/或特定物質表現的條件。此適當條件可為一般 的細胞培養條件，且熟悉此技藝人士自可依照不同的需求 而選擇不同的培養條件。在一實施例中，此適當條件為動 物細胞的培養條件，較佳為CHO細胞的培養條件。在一實 施例中，此適當條件可為37°C下，含3%至10%CO2及5% 至7% 02的潮濕空氣。Weeks, B. S., Yu, Y. M., Kleinman, Η K., and Reddi, A. H., J. Cell Biol., 119: 1721-1728 (1992)). Among the BMP families, BMP-2 has extensive bone formation ability and is the most thoroughly studied. Recombinant human BMP-2 with osteogenic activity has been shown to be safe for use in the treatment and regeneration of bone. The use of BMP-2 has a major impact on traditional orthopedics, formal surgery, periodontal and craniofacial reconstruction surgery. At present, there are two main methods for obtaining BMP-2, namely, extracting BMP-2 protein from demineralized cortical bone and expressing BMP-2 protein by using a performance system. The method of isolating BMP-2 protein from bone tissue requires a large amount of bone tissue and the yield is very low, only about 40 pg of BMP mixture can be obtained per 40 kg of bovine bone powder (Wozney et al., Science 242 (1988), 1528-1534 ). Furthermore, if the BMP-2 protein is isolated from human bone tissue, there is a moral problem and there is a risk of contamination by the source (Jaku's disease, HCV, etc.). Similarly, isolation of BMP homologous proteins from pigs or cattle also poses a similar risk of contamination. Therefore, at present, mainly the use of recombinant BMP-2 to obtain BMP-2 protein performance methods include eukaryotic expression systems and prokaryotic expression systems. The advantages of prokaryotic performance are simple operation, low cost, and high yield. Prokaryotic cells are unable to express the correct BMP-2 precursor relative to the eukaryotic expression system, and thus it is difficult to form a mature BMP-2 protein. The shortcomings of eukaryotic performance are the need for more southern technology and lower yields. Although eukaryotic expression systems produce active BMP-2 proteins, the yield of eukaryotic expression systems is extremely low. It has been previously reported that the addition of low molecular weight (5,000 1^>¥) and high molecular weight (500,000]\^) dextran sulfate increases the production of BMP-2 protein, but its yield is still insufficient. Therefore, the industry needs a s 4 201247874 method to increase bmp production. SUMMARY OF THE INVENTION The present invention provides a bone morphogenetic protein-supply-cell strain H4 method, comprising knowing that the culture medium-culture medium, 41=:gene's this fine running strain is included =r one: the strain is cultured in one culture : base; 1; ==: gene 'the cell /, f this nutrient group includes dextran sulfate. It is obvious that the above and other objects, features, and advantages can be made finer, and in conjunction with the accompanying drawings, the present invention provides a method for preparing bone-forming protein-2, including The cell strain, which contains the gene for bone morphogenetic protein-2, is cultured in a medium in which the medium contains sulfur thioglycan. The ''cell strain'' as used herein refers to any cell strain for protein expression, including animal cells, lower eukaryotes or prokaryotes, preferably animal cells. Animal cells include, but are not limited to, monkeys COS cells, CHO, field cells, human kidney 293 cells, human epidermal A431 cells, human C〇l〇205 cells, 3T3 cells, CV-1 cells, other transgenic primate cells, normal double-set cells, Native tissue-derived cell strain, native transplanted organism, HeLa cell, mouse L cell, BHK, HL-60, 201247874 U937, HaK, PerC6, NOS or Jurkat cells, etc. cultured in vitro. In an embodiment, 'the present invention The cell strain is preferably a CHO cell. The cell line has a bone morphogenetic protein-2 (hereinafter referred to as BMP-2) gene, and the BMP-2 gene can be a self-derived BMP-2 gene of the cell, or can be transfected. To the exogenous BMP-2 gene or recombinant BMP-2 gene in the cell. The BMP-2 gene may be derived from a human or non-human animal (eg 'mouse, rat, hamster, rabbit or camel, etc.). In an embodiment The BMP_2 gene is a human BMP-2 gene, and The bmp-2 gene is transfected into CHO cells. "Appropriate conditions" as used in the present invention means conditions which promote cell survival, cell growth, and/or expression of a specific substance. The appropriate conditions may be general cell culture conditions. And those skilled in the art can select different culture conditions according to different needs. In an embodiment, the suitable condition is the culture condition of the animal cells, preferably the culture condition of the CHO cells. In an embodiment, This suitable condition may be 3% to 10% CO2 and 5% to 7% 02 humid air at 37 °C.
本發明中所述之培養基特別為用於動物細胞之培養 基。使用於本發明之細胞培養基並無特別限制，可為任何 的動物細胞培養基。動物細胞培養基包括含血清培養基， 以及不含動物成分之培養基，例如，無血清培養基、無蛋 白質培養基、及其類似物。在本發明中較佳使用無血清或 無蛋白質培養基，例如，RPMI 1640培養基、Eagle's MEM 培養基、Dulbecco’s modified MEM (DMEM)培養基、199 培養基、F12培養基、Iscove's modified Du】becco’s培養基 (IMDM)、EX-CELLtm 302 培養基、EX_CELLtm-CD_CHO 6 201247874 及EX-CELLTM 325培養基、CHO培養基、CD-CHOTM培養 基、CD OptiCHOT、養基、CD F〇rtiCHOT〜^養基、BD CHO培養基、及CD DG 44TM培養基、IS CD-CHOTM培養 基及Ultra CHO培養基，或上述經修飾、混合或濃縮之培 養基，及其類似物，較佳為DMEM培養基、IMDM培養基、 CD-optic CHO™培養基、Ultra CHO™培養基或其類似物。 在細胞培養的過程中，可加入硫酸葡聚酶（Dextran sulfate)以增加BMP-2蛋白的產量。所使用之硫酸葡聚畴並 無特別限制，其分子量可介於5,000至500,000 Mw，較佳 介於 6,500 至 12,000 Mw，或 8,000 至 10,000 Mw。 硫酸葡聚醣的添加量並無特別限制，原則上硫酸葡聚_ 醣濃度與BMP-2蛋白的產量具有濃度依賴性（d〇Se dependent)，換言之，BMP-2蛋白的產量會隨著硫酸聚_濃 度的增加而增加。培養基中硫酸葡聚醣的濃度可為Oj pg/ml以上，較佳為10 pg/ml以上，更佳為200 gg/ml以上， 最佳介於500至1,000 pg/ml。 本發明另提供一種增加骨形成蛋白-2產量的方法，包 括提供一細胞株，其含有骨形成蛋白-2的基因，將該細胞 株於一適當的條件下培養於一培養基中，其中該培養基包 括硫酸葡聚醣。 在一實施例中，硫酸葡聚醣可增加BMP-2蛋白1倍以 上的產量，較佳為1 -5倍。 【實施例】 1.宿主表現細胞 201247874 本實施例之宿主表現細胞使用缺少二氫葉酸還原酶的 中國倉鼠卵巢細胞(CHO dfhr-)，其購買自台灣食品工業發 展研究所的生物資源保存及研究中心（CCRC, 60133)。將 CHO dfhr-細胞培養於 IMDM 培養基（Isocoves Modified Dulbecco’s Medium IMDM; Gibco Cat. 12200-36)中，其中 含有10%的胎牛血清（Gibco Cat. 10091148)、次黃嘌呤與胸 腺。密咬（Gibco，Cat. 11067-030)、以及 2 mM 的 L-麵醯胺 (Gibco Cat. 25030-081)。Dhfr㈠標誌被用來表現及篩選。藉 由使用二氫葉酸還原酶抑制劑“曱氨蝶呤 (methotrexate，MTX)”（Sigma Cat no. M8407)使 dhfr 基因 表現。當dfhr基因表現時，其他鄰近基因也被一起表現， 選擇表現量高的細胞株，將細胞培養於37°C，5%C02的培 養箱中（Model 3326, Forma scientific)。 2. pND/BMP2表現載體的構築 利用PCR，以全長人類cDNA(LIFESEQ Cat no.IHS1380-97652076)，為模板複製出人類BMP-2基因片 斷，並將BMP-2基因片斷插入pcDNA3.1/Neo (+)/DHFR載 體，形成PND/BMP2載體。此表現載體包含作為篩選標誌 的抗新黴素基因（neomycin-resistance gene) 〇 3. 重組細胞株的建立 將含有人類BMP-2基因的載體以電脈衝法（PA4000The medium described in the present invention is particularly a medium for use in animal cells. The cell culture medium to be used in the present invention is not particularly limited and may be any animal cell culture medium. The animal cell culture medium includes a serum-containing medium, and an animal component-free medium, for example, a serum-free medium, a protein-free medium, and the like. Preferably, serum-free or protein-free medium is used in the present invention, for example, RPMI 1640 medium, Eagle's MEM medium, Dulbecco's modified MEM (DMEM) medium, 199 medium, F12 medium, Iscove's modified Du, becco's medium (IMDM), EX- CELLtm 302 medium, EX_CELLtm-CD_CHO 6 201247874 and EX-CELLTM 325 medium, CHO medium, CD-CHOTM medium, CD OptiCHOT, nutrient, CD F〇rtiCHOT~^, BD CHO medium, and CD DG 44TM medium, IS The CD-CHOTM medium and the Ultra CHO medium, or the above modified, mixed or concentrated medium, and the like, are preferably DMEM medium, IMDM medium, CD-optic CHOTM medium, Ultra CHOTM medium or the like. During cell culture, Dextran sulfate can be added to increase the production of BMP-2 protein. The glucosyl sulfate domain to be used is not particularly limited and may have a molecular weight of from 5,000 to 500,000 Mw, preferably from 6,500 to 12,000 Mw, or from 8,000 to 10,000 Mw. The amount of dextran sulfate added is not particularly limited. In principle, the concentration of glucosyl sulphate and the yield of BMP-2 protein are concentration dependent (d〇Se dependent), in other words, the yield of BMP-2 protein varies with sulfuric acid. The concentration of poly_ increases as the concentration increases. The concentration of dextran sulfate in the medium may be Oj pg/ml or more, preferably 10 pg/ml or more, more preferably 200 gg/ml or more, and most preferably 500 to 1,000 pg/ml. The present invention further provides a method for increasing the production of bone morphogenetic protein-2, comprising providing a cell strain comprising a gene for bone morphogenetic protein-2, and culturing the cell strain in a medium under a suitable condition, wherein the medium is cultured in a medium Including dextran sulfate. In one embodiment, dextran sulfate increases the yield of BMP-2 protein by a factor of more than one, preferably from 1 to 5 times. [Examples] 1. Host expression cells 201247874 The host expression cells of this example used Chinese hamster ovary cells (CHO dfhr-) lacking dihydrofolate reductase, which were purchased from the Taiwan Food Industry Development Institute for conservation and research of biological resources. Center (CCRC, 60133). CHO dfhr- cells were cultured in IMDM medium (Isocoves Modified Dulbecco's Medium IMDM; Gibco Cat. 12200-36) containing 10% fetal calf serum (Gibco Cat. 10091148), hypoxanthine and thymus. Bite (Gibco, Cat. 11067-030), and 2 mM L-face decylamine (Gibco Cat. 25030-081). The Dhfr (a) logo was used for performance and screening. The dhfr gene was expressed by using the dihydrofolate reductase inhibitor "methotrexate (MTX)" (Sigma Cat no. M8407). When the dfhr gene is expressed, other adjacent genes are also expressed together, and a cell strain having a high expression amount is selected, and the cells are cultured in a culture cabinet at 37 ° C, 5% CO 2 (Model 3326, Forma scientific). 2. Construction of pND/BMP2 expression vector The full-length human cDNA (LIFESEQ Cat no. IHS1380-97652076) was used as a template to replicate the human BMP-2 gene fragment and the BMP-2 gene fragment was inserted into pcDNA3.1/Neo. (+)/DHFR vector to form a PND/BMP2 vector. This expression vector contains a neomycin-resistance gene as a screening marker. 3. Establishment of a recombinant cell strain. The vector containing the human BMP-2 gene is subjected to an electric pulse method (PA4000).
PulseAgile® electroporator，Cyto Pulse Sciences)轉染至 CHO dhfr-細胞内。首先將細胞以胰蛋白酶處理後，將細胞 § 8 201247874 懸浮於CP-Τ緩衝液(Cyt〇 pluse Cat. CP-Τ)中使細胞密度為 3父106〇6115/111卜將20(^1的細胞懸浮液（6\105細胞）與10吨 的pND/BMP2混合後進行電脈衝處理，將經電脈衝處理之 細胞培養於不含篩選物質之完全培養基（含1〇%胎牛血清 及HT補充物（HT Supplement)之IMDM)使細胞恢復成長。 培養於不含篩選物質之培養基48小時後，將細胞移至含有 IMDM、10%胎牛血清、L-麩醯胺及5 nM曱氨蝶呤之含篩 選物質的完全培養基中。在培養2週後，將細胞株移至96 孔盤中，並利用ELISA定量分析篩選出高表現rhBMP-2的 細胞株。將篩選出的細胞培養於篩選培養基中。將細胞稀 釋至1 cell/ΙΟΟμΙ的濃度後移至96孔盤中培養至生長成細 胞叢。利用ELISA偵測並定量BMP-2蛋白，再依據ELISA 的結果篩選出高產量細胞並逐步增加MTX濃度從0.005、 0.01、0.02、0.05、0.1 至 0·2μΜ。 4.硫酸葡聚醣對ΒΜΡ-2產量的影響 在本實施例中，分別使用分子量為5000 MW(Fluk.a， FL-31404)、8000 MW(Spectrum，DE131)、6500-10000 MW(Sigma，D4911)、12000 MW(Spectrum’DE133)& 500000 MW(Sigma，D8906)的硫酸葡聚醣。首先，將濃度lxl〇5 之實施例1的細胞培養於2.5 ml的IMDM培養基中’分別 加入不同劑量的硫酸葡聚醣使其最終濃度為〇、1〇、20、 50、100、200、300、500、750 及 1,000 ug/ml 後，將細胞 於37°C下，5 % C02的培養箱内培養7天後，收集培養基 進行ELISA分析。參照第1圖，當硫酸葡聚醣的分子量介 201247874 於6,500-〗2,000時，可有效促進BMP-2蛋白的表現，且 BMP-2蛋白的產量會隨著硫酸葡聚醣濃度的增加而增加。 然而，若硫酸葡聚醣的分子量小於8,000或大於12,000時， 則不會對BMP-2蛋白的產量造成明顯影響。此外，比起分 子量5,000 Mw及500,000 Mw的硫酸葡聚醣，分子量介於 6.500- 12,000硫酸葡聚醣更可增加BMP-2蛋白的產量， 6.500- 12,000硫酸葡聚醣所增加BMP-2蛋白的產量比分子 量5,000 Mw及500,000 Mw硫酸葡聚醣所增加BMP-2蛋白 的產量大1倍以上。 另外，進一步選用 6,500-10,000 MW(Sigma，D4911) 及12,000 MW(Spectrum，DE133)的硫酸葡聚_，分析硫酸 葡聚醣在 0、10、20、50、100、200、300、500、750、1，000 ug/ml濃度下時，BMP-2蛋白的產量。參照第2圖，不管 分子量為6,500-10,000 MW或12,000 MW的硫酸葡聚醣， BMP-2蛋白的產量皆會隨著硫酸葡聚醣濃度的升高而增 加，產量約可增加1-4倍。然而，當濃度大於500 ug/ml 後，BMP-2蛋白的產量就無明顯改變。 雖然本發明已以較佳實施例揭露如上，然其並非用以 限定本發明，任何熟習此技藝者，在不脫離本發明之精神 和範圍内，當可作些許之更動與潤飾，因此本發明之保護 範圍當視後附之申請專利範圍所界定者為準。 201247874 【圖式簡單說明】 第1圖顯示硫酸葡聚醣分子量與濃度對BMP-2蛋白產 量的影響。 第2圖顯示分子量為6,500-10,000 MW或12,000 MW 之硫酸葡聚醣濃度對BMP-2蛋白產量的影響。 【主要元件符號說明】PulseAgile® electroporator, Cyto Pulse Sciences) was transfected into CHO dhfr-cells. After the cells were trypsinized, the cells § 8 201247874 were suspended in CP-Τ buffer (Cyt〇pluse Cat. CP-Τ) to make the cell density 3 father 106〇6115/111 b 20 (^1 The cell suspension (6\105 cells) was mixed with 10 tons of pND/BMP2 and then subjected to electrical pulse treatment. The cells treated with electric pulse were cultured in complete medium containing no screening substance (containing 1% fetal bovine serum and HT supplement). The HT Supplement (IMDM) restores the cells to growth. After 48 hours of culture in the medium without the screening material, the cells are transferred to IMDM, 10% fetal bovine serum, L-glutamate and 5 nM pterin In the complete medium containing the screening substance, after 2 weeks of culture, the cell line was transferred to a 96-well plate, and a cell line with high expression of rhBMP-2 was selected by quantitative analysis by ELISA. The selected cells were cultured in a screening medium. The cells were diluted to a concentration of 1 cell/ΙΟΟμΙ and then transferred to a 96-well plate to grow into a cell cluster. BMP-2 protein was detected and quantified by ELISA, and high-yield cells were screened and gradually increased according to the results of ELISA. MTX concentration from 0.005, 0.01, 0.02, 0.05 0.1 to 0·2 μΜ 4. Effect of dextran sulfate on ΒΜΡ-2 yield In this example, molecular weights of 5000 MW (Fluk.a, FL-31404), 8000 MW (Spectrum, DE131), and 6500, respectively, were used. - 10000 MW (Sigma, D4911), 12000 MW (Spectrum'DE 133) & 500000 MW (Sigma, D8906) dextran sulfate. First, the cells of Example 1 at a concentration of lxl 〇5 were cultured in 2.5 ml of IMDM. After adding different doses of dextran sulfate to the final concentration of 〇, 1〇, 20, 50, 100, 200, 300, 500, 750 and 1,000 ug/ml, the cells were incubated at 37 ° C. After culturing for 7 days in a 5 % C02 incubator, the medium was collected for ELISA analysis. Referring to Figure 1, when the molecular weight of dextran sulfate was 201247874 at 6,500-〗 2,000, the performance of BMP-2 protein was effectively promoted. The yield of BMP-2 protein increases as the concentration of dextran sulfate increases. However, if the molecular weight of dextran sulfate is less than 8,000 or greater than 12,000, it will not significantly affect the production of BMP-2 protein. Compared to dextran sulfate with a molecular weight of 5,000 Mw and 500,000 Mw, molecular weight 6.500-12,000 dextran sulfate can increase the production of BMP-2 protein, 6.500-12,000 dextran sulfate increases the yield of BMP-2 protein by increasing the BMP-2 protein by 5,000 Mw and 500,000 Mw dextran sulfate. The output is more than doubled. In addition, 6,500-10,000 MW (Sigma, D4911) and 12,000 MW (Spectrum, DE133) sulphate sulphate were further selected to analyze dextran sulfate at 0, 10, 20, 50, 100, 200, 300, 500, 750. The yield of BMP-2 protein at a concentration of 1,000 ug/ml. Referring to Figure 2, regardless of the molecular weight of 6,500-10,000 MW or 12,000 MW of dextran sulfate, the yield of BMP-2 protein will increase with the increase of dextran sulfate concentration, and the yield can be increased by 1-4 times. . However, when the concentration was greater than 500 ug/ml, there was no significant change in the production of BMP-2 protein. While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application. 201247874 [Simple description of the diagram] Figure 1 shows the effect of molecular weight and concentration of dextran sulfate on BMP-2 protein production. Figure 2 shows the effect of dextran sulfate concentration of 6,500-10,000 MW or 12,000 MW on BMP-2 protein production. [Main component symbol description]