Coding of thermal sensitive toxin gene of bacillus coli, expressing carrier and application thereof
A technology of Escherichia coli and plant expression vector, applied in the field of genetic engineering, can solve the problems of toxicity of expression products, poor adjuvant effect, low expression level, etc. Effect
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Embodiment 1
[0086] [Example 1] Preparation of Escherichia coli thermosensitive toxin gene NLTA and NLTB of the present invention
[0087] According to the coden usage database published on the Internet, it is calculated that plants prefer to use codons, and then use the biological software DNASTAR to adopt the plant-preferred codes while keeping the amino acid sequences of Escherichia coli thermosensitive toxin A and B subunits unchanged reverse-translation, shielding the plant polyadenylation signal sequence (PPSS) (AATAAA, AATAAT) and mRNA degradation sequence (ATTTA) in the gene sequence at the same time, increasing the G+C% of the gene, and avoiding 15-30 A+T%>80% in 1 base pair, avoid 4 or more consecutive A or T in 10 base pairs, avoid more than 5 consecutive A+T or G+C, avoid some enzymes cutting site, while taking into account the formation of complex secondary structures, the design of new NLTA sequences (SEQ ID NO.1), NLTB sequences (SEQ ID NO.1) and NLTB sequences (SEQ ID NO. ...
Embodiment 2
[0089] [Example 2] Construction of efficient plant expression vector pC234ANLTAB and control wild-type expression vector pC234ACKLTAB
[0090] 2.1 Preparation of Escherichia coli DH5a competent cells
[0091] 1) Take a single colony of the recipient bacteria DH5α grown on the LB solid medium, inoculate it in 5ml LB liquid medium, and cultivate it on a shaking table at 37°C overnight (200r / min);
[0092] 2) Transfer 1% of the inoculum to 100ml LB liquid medium, shake at 37°C and 300r / min for about 3hrs, and measure the OD 600 value (0.4-0.6) to detect the growth status of the culture;
[0093] 3) Under sterile conditions, transfer the bacteria to a sterile, ice-precooled 50ml centrifuge tube, place on ice for 10 minutes, and cool the culture to 0°C;
[0094] 4) 4°C, 4000rpm, centrifuge for 10 minutes, recover the bacterial cells, and remove the culture medium;
[0095] 5) Use 10ml of ice-cold 0.1M CaCl 2 Resuspend each cell pellet and place on ice for 30 minutes;
[0096] ...
Embodiment 3
[0143] [Example 3] Detection of protein expression and activity detection in transgenic plants
[0144] 3.1 Extraction of soluble proteins in transgenic plants
[0145] Take 100 mg of transgenic and non-transgenic plant materials of the present invention, grind them into powder with liquid nitrogen, add 100 ul soluble total protein extract TBSP (10 mM Tris-Cl, 0.02% NaCl, 0.001% PMSF, pH 8.0), and grind for 5 min, 4 After standing at ℃ for 20 minutes, pour the homogenate into a centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, and the supernatant is the extract.
[0146] 3.2 Determination of total protein content by Bradford
[0147] Carry out Bradford method to measure protein content according to the method provided in " Guidebook of Molecular Biology Experiments ", the result is the japonicus japonicus protein extract that transfers the plant expression vector pC234ANLTAB of the present invention, its soluble total protein content is 13ug / ul, transforms wild Type E...
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