鸡白痢沙门菌sptP基因缺失株的构建及其免疫保护效力评价

doi:10.13560/j.cnki.biotech.bull.1985.2020-0714

摘要/Abstract

摘要：

旨在评价鸡白痢沙门菌sptP基因缺失突变株对雏鸡的免疫保护效力,研制有效的鸡白痢沙门菌减毒活疫苗。利用λ-red同源重组技术构建鸡白痢沙门菌C79-13株的sptP基因缺失株C79-13ΔsptP,并分析其基本生物学特性;以1.0 × 10⁸菌落形成单位（CFU）的C79-13ΔsptP经口服免疫3日龄雏鸡,对雏鸡的体重与临床症状变化、血清IgG水平及外周血淋巴细胞增殖能力进行测定,并评价其亲本株C79-13攻毒后的免疫保护效力。PCR和测序结果表明成功构建了鸡白痢沙门菌sptP基因缺失株C79-13ΔsptP,该缺失株与其亲本株C79-13的生长特性和生化特性一致,但其毒力显著降低;免疫后,雏鸡的生长性能没有变化,C79-13ΔsptP能够诱导雏鸡产生显著的体液和细胞免疫应答反应;与对照组相比,攻毒后免疫组雏鸡的发病率和死亡率均显著降低。表明鸡白痢沙门菌sptP缺失株可对雏鸡提供有效的免疫保护,具有作为鸡白痢疫苗的潜力。

关键词: 鸡白痢沙门菌, sptP基因, 免疫保护, 疫苗, 雏鸡

Abstract:

It is aimed to evaluate the immunoprotection of sptP deletion mutant of Salmonella Pullorum and develop an attenuated live vaccine candidate for Salmonella Pullorum. A sptP deletion mutant（C79-13ΔsptP）of Salmonella Pullorum was constructed by λ-red recombination technology,and then the biological characteristics and immunoprotection of C79-13ΔsptP were evaluated. Three-day-old chicks were orally immunized with 1.0 × 10⁸ colony forming units（CFU）of C79-13ΔsptP. The body weight and clinical symptoms of the chicks,serum IgG levels and peripheral blood lymphocyte proliferation ability were measured,and the immunoprotection of parent strain C79-13 after challenge was evaluated. The results from PCR and sequencing showed that sptP deletion mutant of Salmonella Pullorum,C79-13ΔsptP,was successfully constructed,the growth and biochemical properties between the sptP deletion mutant and parent C79-13 were consistent;while the virulence of the sptP deletion mutant significantly decreased. The growth performance of the chicken remained the same after immunization,and C79-13ΔsptP induced the chicken to have specific humoral and cellular immune responses. The morbidity and mortality of the immunized chickens after the challenge significantly reduced compared to the control group,indicating that the sptP deletion mutant offered efficient immunoprotection and demonstrated its potential to be developed as a live attenuated vaccine candidate.

Key words: Salmonella Pullorum, sptP gene, immunoprotection, vaccine, chicken

殷俊磊, 张艳芳, 邹凡雨, 潘鹏涛, 段艳红, 仇书兴. 鸡白痢沙门菌sptP基因缺失株的构建及其免疫保护效力评价[J]. 生物技术通报, 2021, 37(2): 122-128.

YIN Jun-lei, ZHANG Yan-fang, ZOU Fan-yu, PAN Peng-tao, DUAN Yan-hong, QIU Shu-xing. Construction and Immunoprotection of sptP Deletion Mutant of Salmonella Pullorum[J]. Biotechnology Bulletin, 2021, 37(2): 122-128.

图/表 7

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引物名称	引物序列（5'-3'）	扩增片段长度/bp
F1	TGAATCAGCAGGAAGCTCTCAAAAACATACTGCAGGAATgtgtaggctggagctgcttc	1 111
F2	CTTACTTTCAGATAGTTCTAAAAGTAAGCTATGTTTTTAatgggaattagccatggtcc	1 111
CX1	CCGATCTGACAAATACTTAC	2 160或1 561或631
CX2	ATCCGAACTACTTTACGC	2 160或1 561或631
H1	TTTGGCGAGGTGCATACC	391
H2	GTGAGGCGTCTTCCAGCA	391

引物名称	引物序列（5'-3'）	扩增片段长度/bp
F1	TGAATCAGCAGGAAGCTCTCAAAAACATACTGCAGGAATgtgtaggctggagctgcttc	1 111
F2	CTTACTTTCAGATAGTTCTAAAAGTAAGCTATGTTTTTAatgggaattagccatggtcc	1 111
CX1	CCGATCTGACAAATACTTAC	2 160或1 561或631
CX2	ATCCGAACTACTTTACGC	2 160或1 561或631
H1	TTTGGCGAGGTGCATACC	391
H2	GTGAGGCGTCTTCCAGCA	391

菌株	攻毒剂量/CFU	死亡数/总数	LD₅₀/CFU
C79-13	1×10⁸	10/10	3.16 × 10⁶
	1×10⁷	9/10
	1×10⁶	1/10
	1×10⁵	0/10
C79-13ΔsptP	1×10⁹	2/10	> 1.00 × 10⁹
	1×10⁸	0/10
C79-13ΔsptP （pBR322-sptP）	1×10⁹	10/10	6.90 × 10⁶
	1×10⁸	9/10
	1×10⁷	6/10
	1×10⁶	1/10
	1×10⁵	0/10
PBS	--	0/10	--

菌株	攻毒剂量/CFU	死亡数/总数	LD₅₀/CFU
C79-13	1×10⁸	10/10	3.16 × 10⁶
	1×10⁷	9/10
	1×10⁶	1/10
	1×10⁵	0/10
C79-13ΔsptP	1×10⁹	2/10	> 1.00 × 10⁹
	1×10⁸	0/10
C79-13ΔsptP （pBR322-sptP）	1×10⁹	10/10	6.90 × 10⁶
	1×10⁸	9/10
	1×10⁷	6/10
	1×10⁶	1/10
	1×10⁵	0/10
PBS	--	0/10	--

组别	免疫后不同天数雏鸡的平均体重/g
组别	5	12	19
免疫组	66.67 ± 0.82	125.68 ± 1.02	193.71 ± 1.49
对照组	65.81 ± 0.94	128.03 ± 1.57	192.86 ± 1.83