海豚链球菌类M样蛋白基因突变株BY-1ΔsimA的免疫保护作用

doi:10.3969/j.issn.1673-8640.2017.07.021

摘要/Abstract

摘要：

目的探讨海豚链球菌类M样蛋白基因突变株BY-1ΔsimA作为减毒活疫苗保护小鼠免受海豚链球菌感染的能力。方法通过同源重组的方法构建海豚链球菌类M样蛋白基因突变株,利用穿梭载体pVE6007温度敏感、可在链球菌中复制的特征,高效率筛选突变株BY-1ΔsimA。将鉴定正确的BY-1ΔsimA经腹腔注射免疫昆明鼠,21 d后,用3倍半数致死剂量的海豚链球菌BY-1进行攻毒,观察15 d,统计动物死亡数。结果经海豚链球菌突变株BY-1ΔsimA免疫后的昆明鼠感染海豚链球菌BY-1后的死亡率为13.3%,即相对保护率（RPS）为77.8%,与免疫海豚链球菌野生株BY-1全菌灭活疫苗获得的RPS（55.5%）之间差异无统计学意义（P>0.05）。结论海豚链球菌突变株BY-1ΔsimA可有效保护昆明鼠免受海豚链球菌感染,是一种较为理想的减毒活疫苗候选物。

关键词: BY-1ΔsimA, 相对保护率, 海豚链球菌, 昆明鼠

Abstract:

Objective To investigate the immune protection of Streptococcus iniae M-like protein mutant strain BY-1 ΔsimA being used as a live attenuated vaccine to protect mice from Streptococcus iniae infection. Methods Allelic exchange mutagenesis was performed to construct Streptococcus iniae M-like protein mutant strain BY-1 ΔsimA. Shuttle vector pVE6007,which was sensitive to temperature,was used and could replicate in Streptococcus,to screen mutant strain BY-1 ΔsimA in a high efficiency. Kunming mice were immunized by the verified mutant strain BY-1 ΔsimA by intra-peritoneal injection,and infected with lethal dose Streptococcus iniae BY-1 after 21 d. The observation lasted for 15 d,and the accumulated death number was counted. Results Only 13.3% mice immunized with Streptococcus iniae mutant strain BY-1 ΔsimA were dead,resulting in 77.8% relative percentage survival（RPS）,which showed no statistical significance for wild type BY-1 whole-cell killed vaccine（P>0.05）,which had 55.5% of RPS. Conclusions Streptococcus iniae mutant strain BY-1 ΔsimA could effectively protect mice from Streptococcus iniae infection,which was an ideal live attenuated vaccine candidate.

Key words: BY-1ΔsimA, Relative percentage survival, Streptococcus iniae, Kunming mice

中图分类号:

R446.5

杨成良, 李从荣. 海豚链球菌类M样蛋白基因突变株BY-1ΔsimA的免疫保护作用[J]. 检验医学, 2017, 32(7): 647-651.

YANG Chengliang, LI Congrong. Immune protection of Streptococcus iniae M-like protein mutant strain BY-1 ΔsimA[J]. Laboratory Medicine, 2017, 32(7): 647-651.

图/表 4

参考文献 21

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引物	序列（5'~3' ）
simA_F	CGCCATATGGATAGTGATAGATTAACCTTGGAAGNdeI
simA_R	CGCCTCGAGTTCTTCCTCTTTGCGTTTACGXhoI
simA_up_F	CGCGTCGACAGTCTGTTTCAAACTTGTCATGSalI
simA_up_R	TGTGTTTTATATTTTTCTCGTTCATGTTTAGGGTTCTCCTTATTTTC
simA_down_F	AATAGCTATAAATTATTTAATAAGTAAGCTTTCCCTTGCAACCTTTT
simA_down_R	CGCGGATCCAAGTACAAGGATGCTAGCCCTGBamHI
pVE6007_F	TAATACGACTCACTATAGGG
pVE6007_R	TCACTCATCATGTTCATATTTATCA
erm_F	GGAGTAACATAAATGAACGAGAAAAATATAAAACACAG
erm_R	CGTATCTTTCACTTATAAGAATATTACTTATTAAATAATTTATAGC
ITS_F	GAAGATAGGAAAGAGACGCAGTGTC
ITS_R	CCTTATTTCCAGTCTTTCGACCTTC

引物	序列（5'~3' ）
simA_F	CGCCATATGGATAGTGATAGATTAACCTTGGAAGNdeI
simA_R	CGCCTCGAGTTCTTCCTCTTTGCGTTTACGXhoI
simA_up_F	CGCGTCGACAGTCTGTTTCAAACTTGTCATGSalI
simA_up_R	TGTGTTTTATATTTTTCTCGTTCATGTTTAGGGTTCTCCTTATTTTC
simA_down_F	AATAGCTATAAATTATTTAATAAGTAAGCTTTCCCTTGCAACCTTTT
simA_down_R	CGCGGATCCAAGTACAAGGATGCTAGCCCTGBamHI
pVE6007_F	TAATACGACTCACTATAGGG
pVE6007_R	TCACTCATCATGTTCATATTTATCA
erm_F	GGAGTAACATAAATGAACGAGAAAAATATAAAACACAG
erm_R	CGTATCTTTCACTTATAAGAATATTACTTATTAAATAATTTATAGC
ITS_F	GAAGATAGGAAAGAGACGCAGTGTC
ITS_R	CCTTATTTCCAGTCTTTCGACCTTC