Construction and Immunoprotection of sptP Deletion Mutant of Salmonella Pullorum

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

Abstract

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

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.

Figures/Tables 7

References 20

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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