Vitreoscilla hemoglobin mutant and gene and application thereof
A technology of hemoglobin and Vibrio vitreous, applied in the field of genetic engineering, can solve problems such as not necessarily the best
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Embodiment 1
[0034] The construction of embodiment 1 cloning and expression vector
[0035] Utilize the Vector NTI 10 software of Invitrogen Company, design primer VH1 and VH2 according to the vgb of 1.4kb and its promoter sequence reported in literature Dikshit KL, WebsterDA.Gene, 1988,70:377-386:
[0036] VH1: 5'-GC TCTAGA TTATTCAACCGCTTGAGCGT-3' (SEQ ID No. 3) Xba I
[0037] VH2: 5'-CG GAATTC TATGTTAGACCAGCAAACCA-3' (SEQ ID No. 4) EcoR I
[0038] Using the genome of Vitreoscilla filiformis (Vitreoscilla filiformis) ATCC 43191 as a template, VH1 and VH2 were used as primers to amplify the vgb structural gene. The reaction conditions were: 94°C for 5min, 94°C for 30s, 57°C for 30s, 72°C for 40s, after 30 cycles , 72°C for 5 min. The PCR product was separated by 1% agarose gel electrophoresis, and a fragment around 500 bp was recovered. After being digested with EcoR I and Xba I, it was ligated into pUC18 which had been digested by the same double enzyme to obtain pUC18vgb.
[0039]...
Embodiment 2
[0046] Embodiment 2 utilizes error-prone PCR to obtain vgb gene shuffling material
[0047] Use pYG857 template, VH1 and VH2 as primers to amplify vgb, add MgCl to the PCR system 2 and MnCl 2 , so that Mg 2+ and Mn 2+ The final concentrations were 7.5mmol / L and 0.5mmol / L respectively, and the PCR reaction conditions and product processing were as described in Example 1. The error-prone PCR product was recovered by electrophoresis ( figure 2 ), and double-digested with EcoR I and Xba I, and the digested product was connected to the pYG857 that had been digested by the same enzyme to obtain the mutant gene vector pYG857EP, which was transformed into E.coli DH5α competent cells and spread on the LB agar plate containing ampicillin , incubated overnight at 37°C.
[0048] Randomly pick a single transformant colony from the transformation plate, insert it into a 15ml test tube containing 5ml LB liquid medium, add antibiotics, seal the test tube mouth with a rubber cap, and cul...
Embodiment 3
[0049] The DNA shuffling of embodiment 3vgb gene
[0050] (1) DNA fragmentation reaction
[0051] Using pYG857 and 4 pYG857EP screened by error-prone PCR as templates, use the conditions described in Example 1 to amplify the original gene vgb and mutant gene vgb', recover the target fragment by electrophoresis, and dissolve it in an appropriate volume of ddH 2 O middle. In a 20 μl system, add about 0.5 μg of each of the five recovered products and 0.5 UDNase I, react at 25°C for 14-20min, and inactivate the enzyme at 90°C for 10min. The degree of fragmentation of the product was detected by agarose gel electrophoresis. From image 3 It can be seen that when the digestion reaction is carried out for about 18 minutes, an ideal fragment of 50 bp can be obtained. Cut out the gel block where the fragments are concentrated near 50bp, and recover it with the small fragment DNA rapid recovery kit.
[0052] (2) PCR without primers
[0053] Take 40 μl of small fragment recovery pr...
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