Construction of DNA Vaccines of <i>Staphylococcus aureus SarA</i>, <i>IcaA</i> and Their Fusion Genes and Preliminary Study in Mouse Immune Response

doi:10.13523/j.cb.2001070

China Biotechnology

2020, Vol. 40

Issue (7): 41-50 DOI: 10.13523/j.cb.2001070

Construction of DNA Vaccines of Staphylococcus aureus SarA, IcaA and Their Fusion Genes and Preliminary Study in Mouse Immune Response

CHENG Xu¹,YANG Yu-qing¹,WU Sai-nan¹,HOU Qin-long^1,²,LI Yong-mei^1,^2,^**(

),HAN Hui-ming^1,^2,^**(

)

1 Medical College of Beihua University, Jilin 132013, China
2 Center for Infection and Immunity Medical College of Beihua University, Jilin 132013, China

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Abstract

Objective: To investigate the effects of DNA vaccines on the fusion genes of SarA-Azami Green, IcaA-Azami Green, and IcaA-SarA-Azami Green (subsequently Azami Green was represented by AG) and their immune responses in mice.Method:Using the genome of S.aureus as a template, a series of reactions including SarA-AG, IcaA-AG, and IcaA-SarA-AG fusion DNA vaccines were constructed through a series of reactions such as PCR amplification, digestion, and ligation. After transfection into HeLa cells, the transient expression of DNA vaccine was observed under a fluorescent microscope. The cells after two weeks of successful transfection were subjected to genome extraction, and the integration of plasmids on chromosomes was detected by PCR. Recombinant plasmids were extracted using endotoxin-free plasmid extraction kits. BALB / c mice were immunized with AG, SarA-AG, IcaA-AG, and IcaA-SarA-AG. ELISA kits were used to detect mouse IgG antibodies, IL-2, IL-4, IL-13, IFN-γ and TNF-α secretion.Results: SarA-AG, IcaA-AG and IcaA-SarA-AG fusion gene DNA vaccines were successfully constructed. The transfection of the fusion gene DNA vaccine was observed under a fluorescence microscope, and the results showed green fluorescence. Genomic PCR results of the extracted cells showed that the target gene band did not appear. After immunizing mice, SarA-AG, IcaA-AG and IcaA-SarA-AG were able to induce mice to produce higher levels of IgG antibodies. Compared with the blank group and the empty vector AG group, the SarA-AG and IcaA-AG groups were able to secrete higher levels of IL-2 (P<0.001), IFN-γ (P<0.001), and TNF-α (P< 0.001), IL-4 (P<0. 01) and IL-13 (P<0. 01), while IcaA-SarA-AG group TNF-α (P<0.05), IL-4 (P<0. 05)) And IL-13 (P<0.05) were less secreted, but the difference was statistically significant. The secretion of cytokines increased significantly with the increase in the number of immunizations. There was no significant difference in cytokines between the blank group and the empty vector AG group.Conclusion: The DNA vaccines of SarA-AG, IcaA-AG and IcaA-SarA-AG fusion genes were successfully constructed and successfully expressed in eukaryotic cells. PCR verification results confirmed the safety of the DNA vaccine. DNA vaccine can induce humoral immune response and Th1 cell-based cellular immune response after immunizing mice, which has a good application prospect.

Key words： DNA vaccine Eukaryotic expression Immune effect

Received: 21 January 2020 Published: 13 August 2020

ZTFLH:

Q812

Corresponding Authors: Yong-mei LI,Hui-ming HAN E-mail: huiminghan@hotmail.com

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	Xu CHENG
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	Sai-nan WU
	Qin-long HOU
	Yong-mei LI
	Hui-ming HAN

Cite this article:

CHENG Xu,YANG Yu-qing,WU Sai-nan,HOU Qin-long,LI Yong-mei,HAN Hui-ming. Construction of DNA Vaccines of Staphylococcus aureus SarA, IcaA and Their Fusion Genes and Preliminary Study in Mouse Immune Response. China Biotechnology, 2020, 40(7): 41-50.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2001070 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I7/41

Table 1 Primer sequences

Table 2 SarA gene amplification system

Fig.1 SarA,IcaA,IcaA-SarA gene PCR results M: DL2000;1: SarA gene;2: IcaA gene;3: IcaA-SarA gene

Fig.2 PCR results of T vector recombinant plasmid (a)M: DL2000; 1: Negative control; 2: Positive control; 3-4: SarA-T positive clones (b)M: DL2000; 1: Negative control; 2: Positive control; 3-4: All are IcaA-T positive clones (c) M: DL2000;1: Negative control; 2: Positive control; 3-4: All IcaA-SarA-T positive clones

Fig.3 Identification results of T vectoer recombinant plasmid by double enzyme digestion (a) M: DL2000; 1, 3: SarA-T undigested plasmid; 2, 4: SarA-T double-digested plasmid positive results (b) M: DL2000; 1, 3: IcaA-T undigested plasmid; 2, 4: IcaA-T double-digested plasmid positive results (c) M: DL2000; 1, 3: IcaA-SarA-T undigested plasmid; 2, 4: IcaA-SarA-T double-digested plasmid result (2 lane negative result, 4 lane positive result)

Fig.4 T vector recombinant plasmid sequencing results (a) SarA-T sequencing map (b) IcaA-T sequencing map (c) IcaA-SarA-T sequencing map

Fig.5 Identification results of eukaryotic recombinant plasmid by double enzyme digestion (a) M: DL2000; 1, 3: SarA-AG undigested plasmid; 2, 4: SarA-AG double-digested plasmid positive results (b) M: DL2000; 1, 3: IcaA-AG undigested plasmid; 2, 4: IcaA-AG double-digested plasmid positive results (c) M: DL2000; 1, 3: IcaA-SarA-AG undigested plasmid; 2, 4: IcaA-SarA-AG double-digested plasmid

Fig.6 Cells transfection results of DNA vaccine (a)-(c) Field images of SarA-AG, IcaA-AG, and IcaA-SarA-AG fluorescence microscopes (20×);(d)-(f) Fluorescence pictures under SarA-AG, IcaA-AG, IcaA-SarA-AG fluorescence microscope (20×)

Fig.7 Results of DNA vaccine safety by PCR testing M: DL2000; 1, 3, 5: PCR results S. aureus genome as template;2, 4, 6: PCR results using genome-transformed HeLa cells successfully as template. The order is: SarA gene, IcaA gene, IcaA-SarA gene

Fig.8 Detection of mouse serum IgG antibodies *** P<0.001 vs Blank group;;### P<0.001 vs AG group

Fig.9 Detection of mouse serum cytokines (a)IFN-γ (b)IL-2 (c)TNF-α (d)IL-4 (e)IL-13 secretion levels * P<0.05; ** P<0.01; *** P<0.001 vs blank group. # P<0.05; ## P<0.01; ### P<0.001 vs AG group


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