Constructing bicistronic eukaryotic expression plasmid containing human bone morphogenetic protein-2

Constructing bicistronic eukaryotic expression plasmid containing human bone morphogenetic protein-2 labeled with green fluorescent protein*☆

Miao Jun1, Liu Chun-rong2, Huang Hong-chao1, Xia Qun1, Shi Ke-song1, Cui Guo-sheng3

Abstract
BACKGROUND:Bone morphogenetic protein-2 (BMP-2) production of targeted cells is promoted by transfection of adenoviral vectors containing gene, but there are some immune responses. Transfection with plasmid as vector holds promise.
OBJECTIVE: To explore the feasibility to construct human bone morphogenetic protein-2 eukaryotic expression vector labeled with green fluorescent protein (GFP).
DESIGN: Single sample observation.
SETTING: Tianjin Hospital.
MATERIALS: The experiment was performed at the Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2?-T Easy by Dingguo, China; restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.
METHODS: With pcDNA3.1/CT-hBMP2 as template, hBMP2 target fragment was subcloned by PCR binding with designed specific primers. The fragment was bound with pTA2-T-easy and pSELECT-GFPzeo-MCS, separately, and transfected into DH5α cells. pSELECT-GFPzeo-hBMP2 containing GFP was obtained after screening.
MAIN OUTCOME MEASURES: hBMP2 sequence was identified by PCR; whether hBMP2 was cloned into pTA2-hBMP2 and pSELECT-GFPzeo-MCS was identified by digestion and sequencing.
RESULTS: A target fragment of 1 216 bp was obtained by PCR amplification, and cloned into pTA2-T-easy and pSELECT-GFPzeo-MCS. The screening and sequencing results showed that the target fragment was 100% matched with BMP2cDNA sequence (NM-001200) from GenBank.
CONCLUSION: hBMP2 eukaryotic expression vector labeled with green fluorescent protein is successfully constructed.

INTRODUCTION

Bone morphogenetic protein (BMP) was firstly found in the osteogenic research of decalcified bone matrix by Urist [1] in 1965.
As one member of transforming growth factor-β (TGF-β) superfamily, bone morphogenetic protein-2 can induce osteogenic and chondrogenic differentiation and proliferation of mesenchymal stem cells (MSCs), and improve new-bone formation [2-3].
To date, more than 40 kinds of bone morphogenetic protein-2 have been found, and bone morphogenetic protein-2 exhibits the highest osteogenic activity [4].
However, because bone morphogenetic protein-2 is so rare in tissues that bone morphogenetic protein-2 production by gene engineering technique is necessary. In this study, we selected bicistronic eukaryotic expression vector pSELECT-GFPzeo- MCS containing enhanced green fluorescent protein (EGFP) to trace human bone morphogenetic protein-2 expression in vivo.
Moreover, bicistronic eukaryotic expression system enables human bone morphogenetic protein-2 to be expressed highly and stably in animal cells [5].
This study may lay a foundation for further animal experiment of tissue engineered MSCs.

Materials
The experiment was performed at Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007.
pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2?-T Easy by Dingguo, China; competence DH5α Pfu DNA polymerase, Taq polymerase, DNA Marker DL2000, plasmid small extract kit, T4 DNA ligase, plasmid large extract kit, and HRP-DAB substrate coloring kit by Tianjin Runtai; 1Kb DNA Marker and DNA fragment rapid filters recovery by Takara Biotechnology (Dalian); restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.

Methods
Construction of pTA2-hBMP2 cloning vector
One bead of DH5αstrains containing pcDNA3.1/CT-hBMP2 was selected and seeded onto ampicil (Amp)+ solid LB culture plate at 37 ℃ for
overnight. One colony was selected and seeded onto Amp(+) liquid LB, 250 r/min at 37 ℃ water bath for overnight. The plasmids were extracted and electrophoresed on 0.8% agarose gel according to plasmid extract kit introduction. The two sites were selected and designed as BamHI and NheI sites in the upstream and downstream primers of target genes according to characteristics of restriction enzyme of pSELECT-GFPzeo-MCS. Primer was designed by the principles: BamHI site (GGA TCC) and NheI site (GCT AGC) were introduced into KOZAK sequence (ACA ATG G) and two terminal codons (TCA CTA).
Primer sequence was as follows: upstream primer: 5'- ACG GGA TCC ACA ATG GTG GCC GGG ACC CG -3'; downstream primer: 5'- ATA GGC TAG CTC ACT AGC GAC ACC CAC AAC -3'. With pcDNA3.1/CT-hBMP2 recombinant plasmid as template, hBMP2 targeted fragment was amplified by PCR: 4 mL (about 400 ng) plasmids, 10×pfu Buffer (MgCL2 10 mmol) 10 mL, 8 mL dNTP Mixture (2.5 mmol/L), 0.4 mL upstream primer and downstream primer, 0.8 mL Pfu DNA polymerase, and ddH2O. Total was 100 mL. PCR reaction was performed by 30 cycles of predenaturation at 95 ℃ for 5 minutes, denaturation at 94 ℃ for 30 seconds, redenaturation at 58 ℃ for 1 minute and extension at 72 ℃ for 1 minute, then extension at 72 ℃ for 5 minutes and 4 mL DNA polymerase at 72 ℃ for 30 minutes. The amplified PCR products were electrophoresed in 0.8% agarose gels.
The retrieved and purified targeted fragments by PCR product kit introduction were cloned into 1 mL linear pTA2?-T-easy plasmid, 6 mL hBMP2 cDNA, 1 mL 10×T4 Ligase buffer, 1 mL T4 Ligase, and 1 mL rATP at 4 ℃ overnight. The 10 mL mixed system was transfected into DH5α cells and coated on LB culture plate containing X-gal and IPTG overnight. The white colonies were removed. The successfully transfected colonies were cultured at 37 ℃ overnight. The bacterial solution was collected, and plasmids were extracted by alkaline lysis method, identified by BamHI/NheI digestion, and sequenced by Augct, Beijing. Strain was preserved at –80 ℃.

Construction of pSELECT-GFPzeo-hBMP2 vector
pTA2-hBMP2 was digested by NheI and BamHI, and DNA fragments were retrieved. pSELECT-GFPzeo-MCS expression vector was processed as described above at 4 ℃ overnight with T4 DNA ligase. The products were transfected into DH5α cells and coated on LB culture plate containing X-gal and IPTG overnight. The white colonies were removed. The successfully transfected colonies were cultured at 37 ℃ overnight. The bacterial solution was collected, and plasmids were extracted by alkaline lysis method, identified by BamHI/NheI digestion, and electrophoresed in 0.8% agarose gels to detect whether the digested fragments accorded with expectation.

RESULTS

hBMP2 PCR
hBMP2DNA was amplified by PCR, 1 216 bp using pcDNA3.1/CT-rhBMP2 as template, and was found consistent with the expected fragments (Figure 1).

Identification of recombinant plasmid pTA2-hBMP2
Digestion results showed that BamHI/NheI digested products were electrophoresed in 0.8% agarose gels and one fragment of 1 216 bp was found (Figure 2). No bands were found in the other negative control or single enzyme digestion result.

The bacterial solution containing pTA2-hBMP2 recombinant plasmid was sequenced by Augct, Beijing. The sequencing results completely accorded with that of BMP2 (NM-001200) from GenBank.

pSELECT-GFPzeo-rhBMP2 was digested by BamHI/NheI, and the digested products were electrophoresed in 0.8% agarose gels, in which one fragment of 1 216 bp was found. The band was clear with no specific bands.

Sequencing
The bacterial solution containing pSELECT-GFPzeo-rhBMP2 was sequenced by Augct, Beijing. The sequencing results completely accorded with that of BMP2 (NM-001200) from GenBank.

hBMP2 chromosome is located at p21 region of the 20th chromosomes. bone morphogenetic protein-2 has hydrophobic amino acids and secretory protein at N-terminal region, 4 glycosylation sites, and 7 conservative structural domains of aminothiopropionic acid. It plays a role in formation of intrachain disulfide bond, and dimeric preprotein by binding two monomers.
C-terminal mature peptide consisting of 114 amino acid residues is obtained by processing open reading frame (ORF), 1 188 bp, encoding with 396 precursor protein of amino acids, and protease digestion of N-terminal signal peptide and propeptide in center. The conservative structure of mature peptide is folded through 7 pairs of disulfide bonds to form monomers. The dimmer is formed by binding two hBMP2 monomers and secreted out of cells. Homo or heterodimer of mature peptide has bioactivity [6-7].
Bone morphogenetic protein-2 is considered as the highest osteogenic inductor in bone morphogenetic protein-2 family. Bone morphogenetic protein-2 gene can be transfected into cells by gene cloning, and Bone morphogenetic protein-2 growth factor can trigger a series of cell reactions such as chemotaxis of mesenchymal stem cells, induction of mesenchymal stem cells to differentiate into osteoblast and chondrocyte, reconstruction of calcified cartilage, bone tissue formation, and medullary cavity recanalization [8-11].
Transgenic cell transplantation can enhance osteoblast quantity and osteogenic activity, improve bone reconstruction, promote bone formation, and treat osteoporosis, bone defect, bone nonunion, bone tumor, spinal union and other disease [12-15].
Local gene therapy can induce local tissue and cells to express target genes, maintain secretion function, and help bone repair. In this study, we successfully construct pSELECT-GFPzeo-rhBMP-2 vector that can be effectively expressed in eukaryocyte, and lay a foundation for study of rhBMP-2 expression in bone marrow-derived mesenchymal stem cells and tissue-engineered cell transplantation.
Gene technique and expression vector selection are key factors for stable and efficient expression of bone morphogenetic protein-2 during bone repair. To date, many researches have been conducted to transfect target cells with adenovirus vector encoding bone morphogenetic protein-2 gene, and the results demonstrated Bone morphogenetic protein-2 protein secretion. However, further studies have suggested that although adenovirus displays high transfection rate, there are some immune response reactions, which may lead to risk for in vivo experiment. Plasmid as vector can decrease these side effects, and transfection by liposome can achieve 80% transfection rate. In addition, the safety of plasmid, compared with adenovirus vector, makes it suitable for clinical application [16-17].
In our study, we selected pSELECT-GFPzeo-MCS containing GFP to ensure location and tracking of rhBMP2 expression in vivo and vitro because of the characteristics of the expression vector.
pSELECT-GFPzeo-MCS, a eukaryotic bicistronic vector, has strong reproduction that can ensure the stable expression of target gene.
pSELECT-GFPzeo-MCS has two expression cassettes. The expression cassette inserted by exogenous gene contains compound enhancer composed of terminal repeat of elongation factor-1α (EF-1α) and human t-cell leukemia virus[18-19]. SV40 PolyA signal peptide as termination signal ensures the high-level expression of exogenous gene inserted in multiple clone sites [20]; the other cassette contains CMV enhancer. EM7 bacterial enhancer can ensure vector normal expression in prokaryotic cells; β globulin PolyA signal peptide as termination signal contributes to GFP steady transcription and zeo resistance stability [4]; GFP gene is a specific gene, GFP protein and target protein are not fusion protein but independent protein with specific spatial structure and physiological activity.
Zeo resistance gene in pSELECT-GFPzeo-MCS assists Zeo antibiotics to screen target cells containing pSELECT-GFPzeo-MCS. In this study, pSELECT-GFPzeo-rhBMP eukaryotic expression vector was successfully constructed. It can secret bone morphogenetic protein-2 protein in eukaryocyte, and trace target gene expression in vivo and vitro with GFP as report gene.

1 Urist MR. Bone: formation by autoinduction. 1965. Clin Orthop Relat Res 2002; (395):4-10
2 Zachos TA, Shields KM, Bertone AL. Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6. J Orthop Res 2006;24(6):1279-1291
3 Toh WS, Liu H, Heng BC, et al. Combined effects of TGFbeta1 and BMP2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation. Growth Factors 2005; 23(4):313-321
4 Luu HH, Song WX, Luo X, et al. Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res 2007;25(5):665-677
5 Yu J, Russell JE. Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA. Mol Cell Biol 2001; 21 (17):5879-5888
6 Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: molecular clones and activities. Science 1988; 242(4885):1528-1534
7 Tabas JA, Zasloff M, Wasmuth JJ, et al. Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3. Genomics 1991;9(2):283-289
8 Liu HW, Chen CH, Tsai CL, et al. Heterobifunctional poly(ethylene glycol)-tethered bone morphogenetic protein-2-stimulated bone marrow mesenchymal stromal cell differentiation and osteogenesis. Tissue Eng 2007;13(5):1113-1124
9 Yue B, Lu B, Dai KR, et al. BMP2 gene therapy on the repair of bone defects of aged rats. Calcif Tissue Int 2005;77(6):395-403
10 Dai KR, Xu XL, Tang TT, et al. Repairing of goat tibial bone defects with BMP-2 gene-modified tissue-engineered bone. Calcif Tissue Int 2005;77(1):55-61
11 Shirasawa S, Sekiya I, Sakaguchi Y, et al. In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells. J Cell Biochem 2006; 97(1):84-97
12 Park J,Gelse K,Frank S,et al.Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells. J Gene MedJ Gene Med 2006;8(1):112-125
13 Ishikawa H, Kitoh H, Sugiura F, et al. The effect of recombinant human bone morphogenetic protein-2 on the osteogenic potential of rat mesenchymal stem cells after several passages. Acta Orthop 2007; 78(2):285-292

14 Zachos T, Diggs A, Weisbrode S, et al. Mesenchymal stem cell-mediated gene delivery of bone morphogenetic protein-2 in an articular fracture model. Mol Ther 2007;15(8):1543-1550
15 Hasharoni A, Zilberman Y, Turgeman G, et al. Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein-2. J Neurosurg Spine 2005; 3(1):47-52
16 Park J, Ries J, Gelse K, et al. Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes.Gene Ther 2003;10(13):1089-1098
17 Tsuda H, Wada T, Yamashita T, et al. Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene. J Gene Med 2005;7(10):1322-1334
18 Kim DW, Uetsuki T, Kaziro Y, et al. Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system.Gene 1990;91(2):217-223
19 Takebe Y, Seiki M, Fujisawa J, et al. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat.Mol Cell Biol 1988;8(1):466-472
20 Carswell S, Alwine JC. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.Mol Cell Biol 1989;9(10):4248-4258

构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体*☆

苗军1，刘春蓉2，黄鸿超1，夏群1，石可松1，崔国盛3（1天津市天津医院脊柱外科，天津市 300211；2天津武警医学院病理学教研室，天津市 200162；3沧州市第二人民医院，河北省沧州市 061000）
苗军☆，男， 1974年生，河北省沧州市人，汉族， 2004年解放军总医院毕业，博士，主治医师，主要从事脊柱外科和生物材料的研究。
国家自然科学基金资助(30500516)*
摘要
背景：腺病毒虽可携带骨诱导形成蛋白2基因转染靶细胞促进其分泌诱导形成蛋白2，但容易出现一些免疫应答反应，以质粒为载体的转染实验应具有良好的前景。
目的：验证构建绿色荧光蛋白标记的人骨诱导形成蛋白2真核表达载体的可行性。
设计：单一样本观察。
单位：天津医院。
材料：实验于2006-03/2007-03在天津医科大学卫生部激素与发育重点实验室（国家级）完成。含完整hBMP2基因片段的pcDNA3.1/CT-人骨诱导形成蛋白2质粒由李曦铭博士惠赠。双顺反子真核表达载体
pSELECT-GFPzeo-MCS为Invivogen公司产品，Zeo购自Invivogen公司。pTA2?-T Easy为鼎国生物技术有限公司产品，限制性内切酶BamHI 和NheI、T4 DNA连接酶（晶美生物工程有限公司），PCR上下游引物合成及测序（北京奥科生物技术有限责任公司）。
方法：以重组质粒pcDNA3.1/CT-人骨诱导形成蛋白2为模板，结合已设计好的特异性引物，采用PCR方法亚克隆出人骨诱导形成蛋白2目的片段，将该片段分别与克隆载体pTA2-T-easy和双顺反子真核表达载体pSELECT-GFPzeo-MCS连接，转化入感受态DH5α细胞中，通过筛选得到含有绿色荧光蛋白的重组表达载体pSELECT-GFPzeo-人骨诱导形成蛋白2。
主要观察指标：采用PCR法鉴定人骨诱导形成蛋白2序列，同时进行酶切及测序鉴定人骨诱导形成蛋白2是否克隆入pTA2-人骨诱导形成蛋白2重组质粒及真核表达载体pSELECT-GFPzeo-MCS中。
结果：PCR获得长度约1 216 bp的目的片段，经与克隆载体pTA2-T-easy和真核表达载体pSELECT-GFPzeo-MCS连接，筛选及序列分析后，证实所插入目的片段与GenBank检索的BMP2cDNA序列（NM-001200）100%匹配。
结论：成功构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体。
关键词：人骨形态发生蛋白2；真核表达载体；绿色荧光蛋白；组织构建
中图分类号: R329.47 文献标识码: A 文章编号: 1673-8225(2008)20-03984-04
苗军，刘春蓉，黄鸿超，群，石可松，崔国盛. 构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体[J].中国组织工程研究与临床康复,2008,12(20):3984-3987
[www.zglckf.com/zglckf/ejournal/upfiles/08- 20/20k-3984(ps).pdf]
(Edited by J. Terrence Jose Jerome/
Su LL/Wang L)