枯草芽孢杆菌β-葡聚糖酶基因的克隆和表达

摘要/Abstract

摘要：

目的：β-葡聚糖已广泛用于保健和临床治疗。通过酶水解的方法得到分子量大小适中的β-葡聚糖，这一研究日益受到重视，但目前国内还没有公开发表的研究报道。

目的：本实验旨在构建高表达β-葡聚糖酶的工程菌。并对该酶活性及热稳定性做出分析，为今后能够利用到临床医学奠定基础。

方法：通过克隆得到枯草芽孢杆菌β-葡聚糖酶的基因，利用分子生物学技术将β-葡聚糖酶基因与表达载体pET28b+构建重组质粒后导入大肠杆菌中构建工程菌株。利用IPTG诱导其表达目的蛋白，通过镍离子螯合树脂纯化得到目的蛋白。

结果与结论：在本研究中，编码枯草芽孢杆菌β-葡聚糖酶的基因被克隆到大肠杆菌中。并能高效的表达。根据酶活力测定发现，该酶在50 ℃-60 ℃之间有较高的酶活性，在50 ℃最高。 β-葡聚糖酶在30 ℃-40 ℃保持较高的热稳定性。初步发现，β-葡聚糖酶的工程菌表达产物能分解它的底物。但是否适用于临床治疗需要进行更多的研究。

关键词: 枯草芽孢杆菌, 大肠杆菌, 质粒, &beta, -葡聚糖酶, 临床医学, 保健, 克隆, 表达, 酶活性, 治疗

Abstract:

BACKGROUND：β-glucan has been widely used for healthcare and clinical treatment. The molecular weight of the reasonable-sized glucan was obtained By the way of enzymatic hydrolysis which is more and more emphasized, but no research reports has been published in china yet.
OBJECTIVE: The experiment aims to build an engineering strain secreting high-expression β-glucanase of which the activity and thermo-stability are analysed later so as to lay a basis on its clinical application.
METHODS: We got the β-glucanase gene of Bacillus Subtilis，make β-glucanase gene and expression veltor pET28b(+) to recombinant plasmid using molecular biology technology and then structure engineered stain in Escherichia coli, using IPTG to induce the expression of the target potein, purificate it by nickel ion chelating resin.
RESULTS AND CONCLUSION: In this research, Gene encoding β-glucanase in Bacillus subtilis was cloned into Escherichia coli and expressed efficiently. The enzymatic assay has found that it has great activity between 50°C and 60°C and highest in 50°C. The relative high-thermostability of β-glucanase under the temperature of 30°C~40°C. It was preliminarily found that β-glucanase expressed by the engineering strain referred was able to get rid of its substrate. But whether it is suitable for the clinical treatment requires more research. The experiment aims to utilize the strain to improve the level of clinical treatment and provide microbial resources.
Keywords: Bacillus subtilis; Escherichia coli; plasmid; β-glucanase; clinical medicine; healthcare; cloning; expression; enzyme activity; treat

孙珂，王亚楠，张亚南，朱文华，程殿林. 枯草芽孢杆菌β-葡聚糖酶基因的克隆和表达[J]. 中国组织工程研究, 2019, 23(在线): 1-4.

Sun Ke, Wang Ya-nan, Zhang Ya-nan, Zhu Wen-hua, Cheng Dian-lin. Cloning and expression of β-glucanase gene from Bacillus subtilis in Escherichia coli[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(在线): 1-4.

图/表（结果） 4

Cloning of the β-glucanase gene and sequence analysis

Cloning of β-glucanase gene, Bacillus subtilis β-glucanase have 729 bp. Using PCR technology to obtain β-glucanase gene about 749 bp from Bacillus subtilis. the PCR product was electrophoresed on gel agarose 1% and analyzed. A 750 bp fragment was obtained and consistent with the expected size(Fig. 1a). After cloning of this β-glucanase gene in the rapid plasmid using EcoRI single enzyme digestion verification, the plasmids bgl-T on gel agarose showed Two bands with the correct expected size(Fig. 1b). After sequencing is consistent with the published β-glucanase gene sequence size.

Construction and identification of expression plasmid

The bgl-T were digested with EcoRⅠ and BamHⅠ, then ligated into the digested pET28b(+) with the same enzymes and named recombinant plasmid bgl-P, Using EcoRⅠ and BamHⅠdigested plasmid bgl-P. It showed Two bands with the correct expected size(Fig. 2), and sequencing confirmed accurate cloning.

Inducing expression and purification of β-glucanase gene

the bgl-P plasmids were successfully transformed into E. coli strain BL21 (DE3). Induction of expression was done using IPTG. SDS-PAGE revealed that the recombinant enzyme was the major protein in the Cytoplasmic. A 27 kDa band (Fig. 3a), determined by a comparison with the molecular-weight marker on the SDS-PAGE gel. Purification of β-glucanase was done successfully(Fig. 3b)

Enzyme activity determination of β-glucanase

Inducing enzyme activity the product were measured. The results showed that the expression product has β-glucanase activity. Determination of enzyme activity and enzyme stability at different temperatures, Determination of the nature of enzyme activity was found at 50 ℃ has the highest activity and in the 45 ℃-60 ℃ temperature range have a higher activity(Fig. 4). The most stable temperature at 30 ℃- 40 ℃, the relative thermal stability of the enzyme between 50 ℃-60 ℃ maintained at above 50%(Fig. 5).

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引言

材料方法

Design Type: Analytical experiment

Time and place: From 2015 March to August completed in Laboratory of Molecular Biology Qingdao University.

strains, plasmids, and reagents

Bacillus subtilis, Escherichia coli DH5 alpha and Escherichia coli BL21 (DE3) were maintained in the microbiology laboratory, Qingdao university. E. coli cells as the host were grown in LB medium [LB; 1% (w/v) tryptone, 1% (w/v) sodium chloride, 0.5% (w/v) yeast extract; pH 7.0] or on LB agar plate at 37°C. The plasmid pCloneEZ-TA-AMP / HC Easy vector (Clone Smarter Beijing) was used for gene cloning. the vector pET 28b+ was used for the expression of β-glucanase gene. The necessary reagents and kits were purchased from CWBIO (Beijing). Barley β-glucan was obtained from Sigma (San Francisco). All chemicals used were analytical grade, and were obtained from standard suppliers.

Molecular cloning of the β-glucanase gene

To obtain the genomic DNA of Bacillus subtilis were performed as described in Satio and miura^[18]. Cloning of β-glucanase gene from the genomic DNA，PCR amplification of the β-glucanase gene was performed using two primers^[19].

forward primer: 5’-ATT GG ATC CAT GCC TTA TCT GAA ACG A-3’, reverse primer:5’-GGC CGT CTA GAT TAT TTT TTT GTA TAG CG-3’. In the forward and reverse primers, the underlined sequences represent the sites of BamHⅠ and XbaⅠ, respectively. the PCR product was electrophoresed on gel agarose 1% and analyzed. the amplified PCR product was purified using the Gel Extraction Kit. the amplified DNA fragment was cloned into the pCloneEZ-TA-AMP/HC easy vector and obtained the plasmid bgl-T and sequenced. The resulting sequences were analyzed by BLAST in GenBank of NCBI website.

Construction and inducible expression of recombinant expression plasmid

Recombinant plasmid bgl-T was obtained using the PCR purification plasmid kit, double digested with EcoRⅠ and BamHⅠ, ligated into EcoRⅠ-BamHⅠ digested pET-28b(+). The plasmids were purified and analyzed by restriction enzyme digestion using EcoRⅠ and BamHⅠ. After evaluation on gel agarose 1%, the purified plasmids were subjected to sequencing. For expression of β-glucanase, The corrected expression vector was transformed into Escherichia coli BL21 (DE3) competent cells^[20]. The cells were cultivated at 37 ℃with shaking at 200 rpm for 4 h(OD600=0.8-1.0) and IPTG then was added to give 1 mM. Samples were evaluated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the expression of bgl-P. BL21 with bgl-P induced without IPTG, BL21 with pET28b(+) and BL21 no pET28b(+) as a control.

Purification and enzyme activity determination of β-glucanase

The strain was identified as positive By SDS-PAGE electrophoresis protein, and it was inoculated into LB medium containing Ampicillin. After IPTG induction, The cultures were centrifuged and sonicated and purification of β-glucanase was performed by Ni-NTA column. The purified β-glucanase was analyzed by SDS-PAGE gel. Determination of enzyme activity and stability using the method DNS at different temperatures^[21-22]. The optimum temperature was determined by performing the standard enzyme assay within a temperatures range of 45-70 ℃. Thermostability was examined by measuring the residual activity after incubating the enzyme at each specified temperature for fixed time intervals.

Main outcome measures: The activity and thermal stability of the enzyme,which is expressed in E.coli and is punfied by nickel ion chelating resin,were determined at different temperatures.