Fusion expression and identification of B cell epitopes of Toxoplasma gondii surface antigens（SAG）1 and 2

Abstract

Abstract:

Objective To construct the prokaryotic expression system for B cell epitopes of Toxoplasma gondii surface antigens（SAG）1 and 2, and analyze their immunoreactivity.Methods The B cell epitope sequences of Toxoplasma gondii SAG1 and SAG2 were designed and synthesized according to the gene sequences of SAG1 and SAG2 published in GenBank.The target gene was subcloned into pET-28a(+), and expressed in Escherichia coli BL21 (DE3).Complete single colonies were then selected from LB medium containing kanamycin.After culture under shaking to an absorbance(A₄₅₀) value of 0.6-0.8, IPTG was added at a final concentration of 1 mmol/L to induce expression of the target gene.To obtain high concentrations of recombinant SAG(rSAG), the rSAG protein was purified using a His fusion protein purfication kit, and the resultant His-tagged purified protein was identified by Western blotting and ELISA.Results of PCR and double-enzyme digestion showed a specific protein band at ~909 bp.As expected, sequencing of the band further confirmed the construction of rSAG.SDS-PAGE revealed the rSAG molecular weight(M_r) of ~50 000.Western blotting showed specific binding of rSAG with a 1 ∶ 100 dilution of T.gondii-positive mouse serum.ELISA results showed a most positive reaction with the dilution of 1 ∶ 1 for recombinant protein and the dilution of 1 ∶ 50 for positive serum.Moreover, reactions between different dilutions of rSAG and different concentrations of T.gondii-positive mouse serum, and a good performance in distinguishing negative from positive serum.Conclusion The constructed recombinant protein rSAG is soluble and has good immunoreactivity.

Key words: Toxoplasma gondii, Antigen epitopes, Recombination sequence, Immune reactivity

CLC Number:

R382.5

Zhao-zhe WANG, Rui XU, Yang HONG, Jiao-jiao LIN, Ke LU, Hao LI, Zhao-guo CHEN, Yao-jun SHI, Si-min WU, Jia-xin JIANG, Jia-jing LI, Chuan-gang ZHU. Fusion expression and identification of B cell epitopes of Toxoplasma gondii surface antigens（SAG）1 and 2[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(6): 575-579.

Figures/Tables 5

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Table 1

References 13

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血清稀释度Serum dilution		重组蛋白不同稀释度（/10 μg·ml^-1）的A₄₅₀值 Recombinant protein dilution （/10 μg·ml^-1）A₄₅₀
血清稀释度Serum dilution		1 ∶ 1	1 ∶ 2	1 ∶ 4	1 ∶ 8
弓形虫阳性血清Toxoplasma-positive serum	1 ∶ 50	1.037 9	0.809 8^a	0.716 4^a	0.639 7^a
	1 ∶ 100	0.857 6^b	0.730 6^ab	0.608 0^ab	0.514 4^ab
	1 ∶ 200	0.757 0^b	0.692 6^ab	0.574 6^ab	0.476 2^ab
	1 ∶ 400	0.719 8^b	0.682 6^ab	0.554 6^ab	0.472 4^ab
	1 ∶ 800	0.701 8^b	0.673 1^ab	0.554 4^ab	0.472 1^ab
阴性血清Negative serum	1 ∶ 50	0.074 7	0.082 3	0.085 7	0.070 6
	1 ∶ 100	0.082 0	0.073 3	0.080 5	0.087 3
	1 ∶ 200	0.078 9	0.097 3	0.083 0	0.072 9
	1 ∶ 400	0.096 2	0.089 4	0.091 5	0.092 7
	1 ∶ 800	0.086 2	0.080 0	0.083 4	0.083 9

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