Application of Pseudomonas fluorescens iron regulatory protein
A Pseudomonas fluorescens and iron-regulated technology, applied in the field of molecular vaccinology, can solve problems such as unclear effects and achieve significant protective effects
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Embodiment 1
[0017] The expression of iron regulatory proteins TfeR and OprF in Pseudomonas fluorescens is regulated by iron conditions
[0018] Pseudomonas fluorescens TSS1 was shaken and cultivated in normal LB medium (control group) or LB medium containing 600uM iron complex 2,2'-dipyridyl (purchased from Sigma, USA) for 2h at a culture temperature of 28°C. Speed 160rpm. Then centrifuge at 5000g at 4°C for 10min to collect the bacteria. The collected bacteria were subjected to fluorescent quantitative PCR to detect the expression of TfeR or OprF genes (see the literature for specific methods: Zhang SR, Zhang L, Sun L. Identification and analysis of three virus-associated TonB-dependent outer membrane receptors of Pseudomonas fluorescens. Dis Aquat Org .2014;110:181-91.). The results showed that in the presence of the iron complex 2,2'-dipyridyl, the expression levels of TfeR and OprF genes were significantly (P<0.01) up-regulated (that is, the mRNA levels of TfeR and OprF were signi...
Embodiment 2
[0021] Preparation of recombinant Pseudomonas fluorescens iron regulatory proteins rTfeR and rOprF
[0022] Step 1) Construction of expression vectors pETTfeR and pETOprF of rTfeR and rOprF:
[0023] The sequences of Pseudomonas fluorescens TfeR and OprF have been reported (GenBank access ionnumber.AFJ59752.1 and AFJ59305.1). The construction of pETTfeR is as follows: Pseudomonas fluorescens TSS1 is used as a template, and the TfeR gene is amplified by primers F1 / R1. The PCR conditions were: 94°C for 60s to pre-denature the template DNA, then 5 cycles of 94°C for 40s, 60°C for 60s, and 72°C for 60s, then 30 cycles of 94°C for 40s, 66°C for 60s, and 72°C for 60s. PCR products were purified with corresponding kits from Tiangen. The expression vector pET259 (see Hu YH, Zheng WW, Sun L. Identification and molecular analysis of a ferritin subunit from red drum (Sciaenopsocellatus). Fish Shellfish Immunol 2010; 28:678-86 for the construction process of pET259) was treated with res...
Embodiment 3
[0031]Application of Recombinant Pseudomonas Fluorescent Iron Regulatory Proteins rTfeR and rOprF as Vaccines
[0032] Step 1) Preparation of adjuvant and vaccine mixture.
[0033] Preparation of adjuvant control solution: mix 5% (mass ratio) NaOH and 5% (mass ratio) Al 2 (SO 4 ) 3 Mix in a 2:5 volume ratio, and centrifuge the mixture at 10,000g for 5 minutes. Suspend the precipitate in PBS to 0.2mg / ml, which is the adjuvant. Mix equal volumes of PBS and adjuvant, which is the adjuvant control solution.
[0034] Vaccine mixture preparation: Dilute rTfeR or OprF purified in Example 2 above to 200ug / ml in PBS, and mix the diluted protein with an equal volume of adjuvant to obtain rTfeR vaccine mixture or rOprF vaccine mixture.
[0035] The composition of the PBS is by weight percentage: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 .12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.
[0036] Step 2) Immunization application of the vaccine. 120 flounder (each weighing a...
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