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Fifty nine B.b strains were selected, which were isolated in Beijing municipality and 12 provinces and autonomous regions in China. In a previous study, multilocus sequence analysis (MLSA) was used to genotype these strains[8]. They were identified to three pathogenic genotypes: B.b.s.s, B.g, and B.a. Because B.g strains are predominant in China, 1 B.b.s.s, 42 B.g and 16 B.a strains were selected in this study (Table 1).
Provinces Number of strains Genotypes Jilin 14 B.g Guangdong 1 B.g Inner Mongolia 8 B.g Shandong 1 B.a Liaoning 1 B.a Guizhou 3 B.a Sichuan 6 B.a Heilongjiang 5 B.g (3); B.a (2) Xinjiang 15 B.g Beijing 3 B.a Hebei 1 B.g Hunan 1 B.b.s.s Note. B.b.s.s, Borrelia burgdorferi (sensu stricto); B.g, Borrelia garinii; B.a, Borrelia afzelii. Table 1. Distribution of 59 strains in different areas of China
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Strains were cultured in Barbour-Stoenner-Kelly medium, collected by centrifuging at 13,000 rpm/min (r = 15 cm), and then heat-inactivated at 100 ℃. DNA obtained by this method was used as a template for amplifying the gene of 66 kD protein. Primer 5 software was used to design the nucleotide sequences of the primers used in this study according to B31 genome sequence. These are as follows: 5′-GAGCTTGTTCCTGGGTTTGA-3′ and 5′-CTTCCGCTGTAGGCTATTTT-3′. Polymerase chain reaction (PCR) was performed in a total volume of 50 μL. The PCR mix contained 25 μL PCR buffer, 20 pmol/L of each primer, 2.5 mmol/L each of four dNTPs, and 1 U DNA Taq Polymerase (Takara). Amplification was performed for 10 min of an initial denaturation at 94 ℃; 35 cycles under the following conditions: denaturation at 94 ℃ for 45 s, annealing at 41 ℃ for 45 s, extension at 72 ℃ for 90 s, and final extension at 72 ℃ for 10 min. When PCR was performed, negative control (reagent only, no DNA) was included. The positive control was 300 ng DNA from the B.b.s.s strain B31, which is the standard strain in United States. The PCR products’ presence and size were determined by electrophoresis on 1.5% agarose gel in Tris-boric acid-EDTA buffer, followed by staining with GoldView. To validate reproducibility, PCRs were performed at least twice.
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An ABI 3730xl DNA analysis was used to determine the sequences of all PCR products. Distances were calculated using the neighbor-joining method. Sequences that contained 235–1,674 bp of P66 were compared using MEGA 5.10 software[22].
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To amplify the coding gene 235–1,674 bp (encoding 79−558 amino acids) of P66, PCR was used. Neighbor-joining method was used to cluster analysis all strains by MEGA 5.1 software based on the amino acid sequence of P66. The five strains of B.a (details in Result 2), namely LIP94-11, FP1, LB20, LB21, and SZ21, were not included in the cluster analysis because of special mutations in the strains.
Clustering results of the 54 isolates were found to be in accordance with the seven locus sequence analysis[8]. In addition, the B.g strains were divided into three subclusters: subcluster 1, subcluster 2, and subcluster 3. Clustering results are shown in Figure 1.
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The P66 gene of B.b.s.s strain CS4 was found to be exactly the same as the corresponding reference sequence of B31 strain and the 15 Xinjiang strains after the P66 gene sequences of all 59 isolates were combined, adjusted, and analyzed. It was represented by XI91-12 in the subcluster 1, which mutated from CAA to TAA codon in the 508 aa position and led to the premature termination of P66 amino acid sequences of these strains. The IM91-13 sequence in subcluster 1 was changed from TTA codon to TAA codon in 479 aa position, which resulted in the premature termination of the amino acid sequence of P66 protein and nonsynonymous mutations in subcluster 2, subcluster 3, JC1-7, and JC2-2 strains of B.g. Table 2shows the detailed information of the nonsynonymous mutations. In B.a strains, special mutations happened in five strains: a base G was inserted at 438 bp in the p66 gene of LIP94-11 strain, which caused termination of the amino sequence at the 172 aa position; bases A and C were inserted at 1 523 bp in the p66 gene of FP1, LB20, LB21, and SZ21 strains, leading to the early termination of the amino acid sequences.
Genotype Strain AA
positionAA
mutationsGenotype Strain AA
positionAA
MutationsB. garinii Subcluster 2 118 M-I B. garinii JC1-7 109 I-V 153 G-R# 118 M-I 208 T-A 221 I-V 221 I-V 267 I-M 276 L-I 276 L-V 348 V-I 360 N-S 401 A-V 361 S-G 435 A-E 435 A-E 490 T-I 445 A-S 491 T-A 464 M-I 492 S-N 490 T-I 494 A-G 495 S-A 495 S-A 505 E-G 500 A-T 508 Q-K 505 E-G 510 I-T 509 A-T JC2-2 118 M-I 510 I-T 221 I-V Subcluster 3 118 M-I 276 L-V 435 A-E 435 A-E 440 T-A 495 S-A 495 S-A 501 G-E 510 I-A 505 E-G 511 T-I 510 I-T Note. *Reference strains: B.b.s.s-B31, B.g-NMJW1, B.a-HLJ01. #The mutation at 153 aa site occured in 6 strains of subcluster 2. Table 2. Nonsynonymous mutations in P66 gene of 42 B.g strains*
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According to the Immune Epitope Database (IEDB), there is one B-cell epitope (No. 63482) in P66 of B31 strain[23]. The corresponding amino acid sequence is located at 454–491 aa.
Isolates of different genotypes were analyzed according to reference strains of different genotypes. Figure 2 shows the specific mutation information in the different sequences. The amino acid sequence information of six isolates in subcluster 2 of the B.g genotype is represented by the JP1 strain and the other five isolates are represented by the JP2 strain. The detailed information is shown by the P66 amino acid cluster analysis in Figure 1. In subcluster 3 of the B.g genotype, JT4 strain represents the amino acid sequence information of four isolates; and XI91-12 represented amino acid sequence variation of 15 isolates. The variation of FP1 strain represented the four isolates (FP1, LB20, LB21, and SZ21) of the B.a genotype.
The B.g and B.a genotype reference strains and isolates was found to insert aspartic acid (Asp, D) in the 356 aa position and alanine (Ala, A) at the 494 aa position
The 490 aa and 491 aa variations of the B.g subcluster 2 strains and the 464 aa and 490 aa variations of JC1-7 strain affect the only one B-cell epitope of the P66 protein antigen.
Polymorphism of P66 in Borrelia Burgdorferi Strains in China
doi: 10.3967/bes2021.048
- Received Date: 2020-08-12
- Accepted Date: 2020-12-04
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Key words:
- Borrelia burgdoferi /
- P66 /
- B-cell epitope /
- Polymorphism
Abstract:
Citation: | HAO Qin, LIU Hui Xin, HOU Xue Xia, ZHANG Lin, YANG Xiao Na, WAN Kang Lin. Polymorphism of P66 in Borrelia Burgdorferi Strains in China[J]. Biomedical and Environmental Sciences, 2021, 34(5): 364-371. doi: 10.3967/bes2021.048 |