和rbFGF对BALB/C3T3成纤维细胞增殖的促有丝分裂作用通过之前描 述的MTT方法进行分析。简要地说，将BALB/C3T3细胞以5X104的细胞密度接种至96孔板 中，在补充有1. 5%胎牛血清的DMEM培养基中37°C培养24小时。然后，分别使用以下混合 物孵育所述细胞培养3天：（1)DMEM和EGF样品；（2)DMEM和商业化的EGF; (3)DMEM和bFGF 样品；（4)DMEM和商业化的bFGF21; (5)DMEM。随后，向每孔中加入20y1的MTT(3-(4, 5-二 甲基噻唑-2基）-2, 5-二苯基四氮唑溴化物）溶液，然后在37°C将平板孵育4小时。将液 体从每个孔中移除，然后加入150y1的DMS0来溶解紫色的晶体。使用酶标仪在570nm波 长下对所述平板进行读数。相同的测试重复4次。由生长的细胞产生的NADPH还原酶活性 造成的MTT的减少导致紫色甲臢的形成（0D570nm下检测），提供了一种有助于量化细胞增 殖的方法。
[0080] 应当理解的是，在独立的实施方式和实验中为了清除而描述的本发明的一些特 征，可以在单个实施方式或实验中进行组合提供。相反的，在单个的实施方式和实验中为了 简洁而描述的本发明的多种特征可以独立提供或者在任何合适的亚组合中提供。值得注意 的是，实施方式或实验的一些特征是通过非限制性实施例来说明的。同样，本领域的技术人 员知晓出于简洁的目的而未列出的现有技术。
[0081] 将以下参考文献的全文引入本申请中作为参考。
[0082] Aliboiandi M, Mirzahoseini H (2011) Purification and refolding of overexpressed human basic fibroblast growth factor in Escherichia coii. Biotechnol Res Int 2011:1-6
[0083] Andrades J A, Santamaria JA, ffu LT,Hall FL, Nimni ME, Becerra J(2001) Production of a recombinant human basic fibroblast growth factor with a collagen binding domain. Protopl asma 218:95-103
[0084] Asubel F, Brent R, Kingston R, Moore D, Seidman j, Smith J, Stroh ! K(1993) Curr Protoc Mol Biol 2:11.2.1-11.2.5
[0085] Ban-PJ，Cousens LS，Lee-Ng CT，Medina-Seiby A，Masiarz FR，Hailewell RA,Chamberlain SH, Bradley JD，Lee D，Steimer KS(1988)Expression and processing of biologically active fibroblast growth factors in the yeast Saccharomyces cerevisiae. J Biol Chem263:16471一16478
[0086] Bikfalvi A，Klein S，Pmtucci G，Rifkm DB (1997)Biological Roles of Fibroblast Growth Factor-2. Endocr Rev 18:26-45
[0087] Chan AKN, Wang YY,Ng KL, FuZ,Wong WKR(2012)Cloning and characterization of a novel celiobiase gene，cba3,encoding the first known旦-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea. Gene 493:52-61
[0088] Cottingham IR，Millar A，Emslie E，Colman A，Schiiieke AE，Me ee C (2001) A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli.Nat Biotechnol 19:974-977
[0089] El ! euche S,Poggeler S (2010) Inteins, valuable genetic elements in molecular biology and biotechnology. Appl Microbiol Biotechnol 87:479-489
[0090] Esipov RS, Stepanenko VN, Chupova LA, Boyar ski kh UA, Filipenko ML, Miroshnikov AI (2008)Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system. Protein Expr Purif 61:1-6
[0091] Ferrer-Miralles N，Domingo-Espin J，Corchero JL,Vazquez E, Viilaverde A (2009)Microbial factories for recombinant pharmaceuticals.Microb Cell Fact 8:17
[0092] Fu ZB, Ng L, Lam TL, Wong WKR(2005)Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli.Protein Expr Purif 42:67-77
[0093] Fu ZB, Ng KL, Lam CC, Leung KC,Yip WH, Wong WKR (2006) A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli. Protein Expr Purif 48:205-214
[0094] Garke G,Deckwer WD, Anspach FB (2000)Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cel1-density cultivation of Escherichia coli.J Chromatogr B Biomed Sci Appl 737:25-38
[0095] Huang RC, Lam E, Chen YH, Hackett J, Lam TL, Liu D, Ma MC, Siu KL, Sivakesava S, Xu ZN, Wong RSC, Wong WKR(1999)Human epidermal growth factor excreted by recombinant Escherichia coli K-12 has the correct'N-terminus and is fully bioactive. Process Biochem 35:1-5
[0096] Ingham AB, Sproat Kff,Tizard MLV, Moore RJ(2005)A versatile system for the expression of nonmodified bacteriocins in Escherichia coli. J Appl Microbiol 98:676-683
[0097] wong WY, Ng L, Lam CC, Wang YY, WongffKR(2013) Authentic human basic fibroblast growth factor produced by secretion in Bacillus subtilis. Appl Microbiol Biotechnoi 97:6803-6811
[0098] Lam TL, Wong RSC, Wong ff R(1997)Enhancement of extracellular production of a Celluiomonas flmi exogiucanase in Escherichia coli by the reduction of promoter strength. Enzyme Microb Techno1 20:482-488
[0099] Lee C，Li P，Inouye H，Brickman ER，Beckwith J (1989) Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J Bacterioi 171:46094616
[0100] Li Y (2011) Self-cleaving fusion tags for recombinant protein production. Biotechnoi Lett 33:869-881
[0101] Mirzahoseini H,Mehraein F,Omidinia E,Razavi MR(2004)Differential expression of human basic f