金黄色葡萄球菌IsdB表位蛋白与HBc蛋白融合表达

doi:10.13481/j.1671-587x.20150212

Abstract

Abstract:

Objective To construct the E.coli expressiom system of the HBc-IsdB_50-285 fusion protein, and to explore the expression efficacy of the HBc-IsdB_50-285 fusion protein in prokaryotic system.Methods HBc-IsdB_50-285 fusion gene fragment was designed and cloned,and the expression vector pET-28-HBc-IsdB_50-285 was construct and transformed into E.coli BL21,the expression of protein was inducted by IPTG. The solubility of protein and its relative molecular mass were analyzed by SDS-PAGE method; BCA assay was used to detect the level of protein; the immune activity of the recombinant protein was detected by immune inoculation method.40 mice were divided into rIsdB+adjuvant group(n=10), HBc-IsdB_50-285+adjuvant group(n=10), HBc-IsdB_50-285 group(n=10), and PBS group(n=10).The values of special IgG of the mice were tested by ELISA method. The mice were immunized and attacked by Stathylococcus aureus Newman strain,and the immune protection rate of the recombinant protein was detected.Results The expression vector pET-28-HBc-IsdB_50-285 was successfully constructed through identification of sequencing and restriction enzyme digestion. The SDS-PAGE results showed that the recombinant protein existed in a soluble form in the supernatant with relative molecular mass about 44 000.The level of purificated protein was 1.0 g·L^-1.The ELISA results showed that the value of antibody titer of the mice in HBc-IsdB_50-285+adjuvant group was lower than that in rIsdB+adjuvant group when the rIsdB were regarded as coating antigen(P<0.01);the values of antibody titer of the mice in HBc-IsdB_50-285+adjuvant group was higer than those in rIsdB+adjuvant group and HBc-IsdB_50-285 group when the HBc-IsdB_50-285 were regarded as coating antigen(P<0.05);the attack test results showed that the protection rate of Staphylococcus aureus of the mice in HBC-IsdB_50-285+adjuvant group was lower than that in rIsdB+adjuvant group (P>0.05). Compared with HBc-IsdB_50-285 group, the protection rate of Staphylococcus aureus of the mice in HBC-IsdB_50-285+adjuvant group had no significant difference (P>0.05). Compared with PBS group, the protection rates of Staphylococcus aureus of the mice in HBc-IsdB50-285+adjuvant group(P<0.01) and HBc-IsdB_50-285 group(P<0.05) were increased.Conclusion A prokaryotic expression vector of HBC-IsdB_50-285 is constructed, and the HBC-IsdB_50-285 fusion protein with better biological activities is expressed in E.coli BL21 successfully.

Key words: Staphylococcus aureus, hepatitis B virus core protein, iron-regulated surface determinant B, epitope

CLC Number:

R378.1

BAI Liang, CHENG Yan, XU Fangfei, LIU Yan, ZHANG Shujun, CAO Ruizhen. Fusion expression of Staphylococcus aureus IsdB epitope protein and HBc protein[J].Journal of Jilin University Medicine Edition, 2015, 41(02): 269-274.

References

[1] Fowler VG Jr, Miro JM, Hoen B, et al. Staphylococcus aureus endocarditis:a consequence of medical progress[J]. JAMA, 2005, 293(24):3012-3021.

[2] Klevens RM,Morrison MA, Nadle J, et al.Invasive methicillin-resistant Staphylococcus aureus infections in the United States[J].J Am Med Assoc,2007,298(15):1763-1771.

[3] Mazmanian SK, Skaar EP, Gaspar AH, et al. Passage of heme-iron across the envelope of Staphylococcus aureus[J]. Science,2003, 299(5608):906-909.

[4] Brown M, Kowalski R, Zorman J, et al. Selection and characterization of murine monoclonal antibodies to Staphylococcus aureus iron-regulated surface determinant B with functional activity in vitro and in vivo[J]. Clin Vaccine Immunol,2009, 16(8):1095-1104.

[5] Ebert T, Smith S, Pancari G, et al. A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo[J]. Hum Antibodies, 2010, 19(4):113-128.

[6] Schodel F, Moriarty AM, Peterson DL, et al. The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity[J]. J Virol,1992, 66(1):106-114.

[7] Roose K, De Baets S, Schepens B, et al. Hepatitis B core-based virus-like particles to present heterologous epitopes[J]. Expert Rev Vaccines, 2013, 12(2):183-198.

[8] 张占东, 徐静, 张云涛, 等. HBcAg-VEGF抗原表位融合蛋白的原核表达、纯化及其免疫原性[J]. 中国生物制品学杂志, 2013, 26(9):1278-1284.

[9] Yin Y, Zhang J, Chen W, et al. Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis[J]. Vaccine, 2008,26(46):5814-5821.

[10] Yin Y, Zhang S, Chen W, et al. Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine[J]. Immunobiology, 2014, 219(2):97-103.

[11] Stranger-Jones YK, Bae T, Schneewind O, et al, Vaccine assembly from surface proteins of Staphylococcus aureus[J]. Proc Natl Acad Sci USA,2006, 103(45):16942-16947.

[12] Botelho-Nevers E, Verhoeven P, Paul S, et al. Staphylococcal vaccine development:review of past failures and plea for a future evaluation of vaccine efficacy not only on staphylococcal infections but also on mucosal carriage[J]. Expert Rev Vaccines, 2013, 12(11):1249-1259.

[13] Etz H, Minh DB, Henics T, et al. Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus[J]. Proc Natl Acad Sci USA,2002, 99(10):6573-6578.

[14] Spellberg B, Daum R. Development of a vaccine against Staphylococcus aureus[J]. Semin Immunopathol,2012, 34(2):335-348.

[15] Bottcher B, Wynne SA, Crowther RA, et al. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy[J]. Nature, 1997, 386(6620):88-91.

[16] Kratz PA, Bottcher B, Nassal M. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids[J]. Proc Natl Acad Sci USA,1999, 96(5):1915-1920.

[17] Block H, Kubicek J, Labahn J, et al.Production and comprehensive quality control of recombinant human interleukin-1beta:a case study for a process development strategy[J].Protein Expr Purif, 2008, 57(2):244-254.

Fusion expression of Staphylococcus aureus IsdB epitope protein and HBc protein

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 5

Metrics

Comments

Recommended 0

[1]	WANG Junrui, DUAN Meiqing, Erdemutu, SUN Peng, WEI Changmei, HAN Yanqiu. Effects of sub-inhibitory concentration of imipenem on proliferation in vitro and mRNA expression levels of MRSA virulence related genes [J]. Journal of Jilin University Medicine Edition, 2017, 43(03): 479-484.
[2]	XIN Guijie, ZHU Haichao, LI Hao, WEN Jianping. Induction of cellular immunity by HCV multi-epitope DNA vaccine prepared with minimalistic immunologically defined gene expression method in mice [J]. Journal of Jilin University Medicine Edition, 2016, 42(06): 1071-1075.
[3]	YANG Si-Rui, ZHAI Chu-Bo, WU Xiu-Li, YU Yong-Li, WANG Li-Ying. Protective effect of fusion protein of hsp65-MOMP-T-epitopes on C. trachomatis genital tract infections in mice [J]. J4, 2010, 36(5): 887-890.
[4]	WANG Yan-shu,TIAN Ya-ping,WAN Min,YU Yong-li,WANG Li-ying. Construction and expression of human papillomavirus CTL epitope vaccine [J]. J4, 2007, 33(2): 260-264.
[5]	YANG Si-rui, WEI Hong-fei，SUN Jing-hui，YANG Yu，WANG Yan-meiLU Ji-rong，YU Yong-li，WANG Li-ying. Gene recombinant and prokaryotic expression of HSP65-T cell epitopes of MOMP of C.trachomatis fusion protein and　its purification [J]. J4, 2006, 32(1): 11-4.