Construction and Phenotypic Analyses of Recombinant Lactobacillus Expressing Single-Chain Antibody of HIV

doi:10.13523/j.cb.20191001

China Biotechnology

2019, Vol. 39

Issue (10): 1-8 DOI: 10.13523/j.cb.20191001

Construction and Phenotypic Analyses of Recombinant Lactobacillus Expressing Single-Chain Antibody of HIV

AN Ming-hui^1,^2,³,TIAN Wen^1,^2,³,HAN Xiao-xu^1,^2,³,SHANG Hong^1,^2,^3,*^*

1 NHC Key Laboratory of AIDS Immunology (China Medical University), The First Affiliated Hospital of China Medical University, Shenyang 110001, China
2 Key Laboratory of AIDS Immunology of Liaoning Province,The First Affiliated Hospital of China Medical University, Shenyang 110001, China
3 Key Laboratory of AIDS Immunology, Chinese Academy of Medical Sciences, Shenyang 110001, China

Download:

HTML

PDF(1177KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Recently, although the passive immunization strategy based on antibody drugs has achieved certain results in the prevention and treatment of HIV, supplement of antibodies is necessary to maintain its long-term effective concentration. The key question is how to prolong the active time of antibodies. Due to the food safety and mucosal colonization stability of lactobacillus, the anti-HIV single-chain antibody fragments were inserted in its expression plasmid with homologous promotor and signal peptide, and plasmid was transformed into competent cells of WCFS1 strains by electroporation. Positive single colonies were screened out by erythromycin, and after culture, the anti-HIV single-chain antibodies were detected to be expressed in the bacterial supernatant and the surpernatant can inhibit the HIV pseudovirus. Compared with the wild-type WCFS1 strain, the growth capacity of the recombinant strain in vitro did not changed and the CFU of the bacterial solution could reach to10 ⁹/ml after 24 hours’ fermentation; the colonization ability of the recombinant strain also did not change obviously, and the each square centimeter of monolayer epithelial cells could bind 10 ⁵CFU colonies. Expressing anti-viral proteins via a live recombinant lactobacillus as host rectal mucosal colonizers gave a new proposal to prevent HIV homosexual transmission.

Key words： Single-chain variable fragment (scFv) of HIV Lactobacillus plantarum Promotor Signal peptide Mucosal epithelial cells

Received: 14 March 2019 Published: 12 November 2019

ZTFLH:

Q815

Corresponding Authors: Hong SHANG

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Ming-hui AN
	Wen TIAN
	Xiao-xu HAN
	Hong SHANG

Cite this article:

AN Ming-hui,TIAN Wen,HAN Xiao-xu,SHANG Hong. Construction and Phenotypic Analyses of Recombinant Lactobacillus Expressing Single-Chain Antibody of HIV. China Biotechnology, 2019, 39(10): 1-8.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191001 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I10/1

Table 1 The list of primers

Fig.1 Modification and identification of expressing plasmid (a)Lane1-2: Anti-ampicillin gene; Lane3: 2 000bp DNA marker (b) Lane2: 10kb DNA marker;Lane 1,4: The bands of plp0373_AmyA(Em/Amp) with and without double enzyme digestion;Lane 3,5: The bands of previous plp0373_AmyA with and without double enzyme digestion (c)Lane4: 2 000bp DNA marker;Lane1-3: Promoter lhdl gene (d) Structure diagram of modified plasmids

Fig.2 The construction of recombinant plasmid (a)Lane1-2:scFv gene targeting HIV envelope protein;Lane3-4:scFv gene targeting CD4 molecular (b)Structure diagram of recombinant plasmids

Fig.3 Western blot analysis of anti-HIV scFv 1: Wild strain;2: scFv1 targeting HIV envelope protein; 3: scFv2 targeting CD4 molecular

Table 2 Neutralizing antibody of the supernatant of two recombinant WCFSs against different HIV-1 pseudoviruses

Fig.4 The neutralizing curve of the supernatant of two recombinant WCFSs against different HIV-1 pseudoviruses Y-axia is viral inhibition rate, X-axia is the dilution of supernatant

Fig.5 The comparison of growing ability in vitro between recombinant and wild WCFS1 strains

Fig.6 The comparison of colonization ability on the mucosal epithelial cells between recombinant and wild WCFS1 strains


[1]	Gunthard H F, Aberg J A, Eron J J , et al. Antiretroviral treatment of adult HIV infection:2014 recommendations of the international antiviral society-USA panel. JAMA, 2014,312(4):410-425.

[2]	Siliciano J D, Siliciano R F . Recent developments in the effort to cure HIV infection: going beyond N=1. J Clin Invest, 2016,126(2):409-414.

[3]	Trovato M , D’Apice L,Prisco A,et al.HIV vaccination:a roadmap among advancements and concerns. Int J Mol Sci, 2018,19(4):1241.

[4]	Sahay B, Nguyen C Q, Yamamoto J K . Conserved HIV epitopes for an effective HIV vaccine. J Clin Cell Immunol, 2017,8(4):518.

[5]	Saunders K O, Pegu A, Georgiev I S , et al. Sustained delivery of a broadly neutralizing antibody in nonhuman primates confers long-term protection against simian/human immunodeficiency virus infection. J Virol, 2015,89(11):5895-5903.

[6]	Caskey M, Klein F, Lorenzi J C , et al. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature, 2015,522(7557):487-491.

[7]	Beyrer C , Baral S D,van Griensven F,et al.Global epidemiology of HIV infection in men who have sex with men. Lancet, 2012,380(9839):367-377. doi: 10.1016/S0140-6736(12)60821-6

[8]	Anbazhagan K, Sasikumar P, Gomathi S , et al. In vitro degradation of oxalate by recombinant Lactobacillus plantarum expressing heterologous oxalate decarboxylase. J Appl Microbiol, 2013,115(3):880-887.

[9]	Mathiesen G, Sveen A, Piard J C , et al. Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides. J Appl Microbiol, 2008,105(1):215-226.

[10]	Sasikumar P, Gomathi S, Anbazhagan K , et al. Genetically engineered lactobacillus plantarum WCFS1 constitutively secreting heterologous oxalate decarboxylase and degrading oxalate under in vitro. Curr Microbiol, 2014,69(5):708-715. doi: 10.1007/s00284-014-0644-2

[11]	Sasikumar P, Gomathi S, Anbazhagan K , et al. Secretion of biologically active heterologous oxalate decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 using homologous signal peptides. Biomed Res Int, 2013,2013:280432.

[12]	Wu X, Guo J, Niu M , et al. Tandem bispecific neutralizing antibody eliminates HIV-1 infection in humanized mice. J Clin Invest, 2018,128(6):2239-2251.

[13]	Ahrne S, Nobaek S, Jeppsson B , et al. The normal Lactobacillus flora of healthy human rectal and oral mucosa. J Appl Microbiol, 1998,85(1):88-94.

[14]	Nazzaro F, Fratianni F, Orlando P , et al. Biochemical traits,survival and biological properties of the probiotic Lactobacillus plantarum grown in the presence of prebiotic inulin and pectin as energy source. Pharmaceuticals (Basel), 2012,5(5):481-492.

[15]	Kleerebezem M , Boekhorst J,van Kranenburg R,et al.Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci USA, 2003,100(4):1990-1995.

[16]	Hols P, Slos P, Dutot P , et al. Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826. Microbiology, 1997,143(Pt8):2733-2741.

[17]	王巧惠, 王光强, 宋馨 , 等. 不同质粒电转三种乳酸菌的比较研究. 工业微生物, 2016,46(1):36-41.

[17]	Wang Q H, Wang G Q, Song X , et al. Comparative studies on electricity transformation of three kinds of lactic acid bacteria using different plasmids. Industrial Microbiology, 2016,46(1):36-41.

[18]	Aukrust T W, Brurberg M B, Nes I F . Transformation of Lactobacillus by electroporation. Methods Mol Biol, 1995,47:201-208.

[1]	HE Ruo-yu,LIN Fu-yu,GAO Xiang-dong,LIU Jin-yi. *Research and Application Progress of Signal Peptides in Escherichia coli* Secretion Systems**[J]. China Biotechnology, 2021, 41(5): 87-93.

[2]	Nan WANG,Lv-hua JIN,Ling ZHANG,Rong LIN,Hai-lin YANG. The Effect of Signal Peptides on the Expression of Leucine Dehydrogenase and Enzymatic Properties in Bacillus subtilis[J]. China Biotechnology, 2018, 38(4): 46-53.

[3]	ZHANG Ling,WANG Nan,JIN Lv-hua,LIN Rong,YANG Hai-lin. *To Promote the Expression of Leucine Dehydrogenase in Bacillus subtilis* via Dual-Promoter and Fermentation Research**[J]. China Biotechnology, 2018, 38(12): 21-31.

[4]	YANG Qing, WANG Bin, WANG Ya-wei, ZHANG Hua-shan, XIONG Hai-rong, ZHANG Li. Comparison of Signal Peptides for Two Hemicellulase Secretory Expression[J]. China Biotechnology, 2017, 37(8): 15-22.

[5]	CHEN Long-guan, QIN Jin-hong, HUANG Yun-na, MAI Jun-xin, XIE Qiu-ling. Optimized Signal Peptides Sequences for the Development of High Expressing McAbs[J]. China Biotechnology, 2016, 36(3): 77-81.

[6]	ZHOU Yong, XU Gang, YANG Li-rong, WU Jian-ping. Effects of Signal Peptides's Optimization on the Secretion of Lipase S in Bacillus subtilis[J]. China Biotechnology, 2015, 35(9): 42-49.

[7]	CHEN Ke, HUANG Xiao, LU Lei, XING Yun, HOU Jing, WU Jie, LIU Jing-jing. *Comparison of celY* Gene from Erwinia chrysanthemi Expression in Escherichia coli Driven by Different Regulatory Elements**[J]. China Biotechnology, 2012, 32(02): 24-28.

[8]	JIA Qi-Yu, WANG Yu-Guang, SUN Jian-Bei, LEI Xue-Hua, GU Wen-Liang. Isolation of a Strain of Endophytic Bacteria against Banana Fusarium Wilt and Activity Analysis of Signal Peptide of its Chitinase[J]. China Biotechnology, 2010, 30(09): 24-30.

[9]	HU E-Dong, TAO Dong-Sheng. Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/α-factor for Saccharomyces cerevisiae[J]. China Biotechnology, 2009, 29(12): 49-53.

[10]	. Analysis and Prediction of Secreted Proteins in Escherichia coli UTI89[J]. China Biotechnology, 2007, 27(7): 100-105.

[11]	. Efficient Secretion of Recombinant PEX in COS7 Cells[J]. China Biotechnology, 2007, 27(5): 1-5.

Viewed

Full text

Abstract

Cited

Shared

Discussed