[0065] Example 1
[0066] 1. HN-VP2 33-221aa Acquisition of fusion protein coding gene sequence
[0067] (1) Acquisition of HN gene of Newcastle disease virus
[0068] According to the nucleotide sequence of the HN gene of Newcastle disease virus in GenBank (No: JF343538), a pair of specific primers were designed to amplify the HN gene fragment of Newcastle disease virus using DNA Star7.0 and Primer premier 6.0 software.
[0069] P1 and P2 amplify the HN gene of Newcastle disease virus, and introduce the 5'end of primer P1 Bam At the HI endonuclease site, a flexible linker was introduced at the 5'end of P2. The primer sequence is as follows:
[0070] Table 1 The primer sequences used in PCR amplification
[0071]
[0072] The frozen NDV strains were thawed in a 37°C water bath and then inoculated with 9-10 day-old non-immunized chicken embryos (0.2 mL per embryo) through the allantoic cavity, and incubated at 37°C. Collect aseptically the allantoic fluid of dead chicken embryos for 24 to 48 hours (the chicken embryo fluid that has red blood cells or egg yolk makes the allantoic fluid turbid is discarded), after repeated freezing and thawing 3 times, centrifuged at 8 000r/min 4℃ for 15min, Take the supernatant and store at -20°C until use. The extraction of viral RNA is performed in accordance with the instructions of the RNA extraction kit. Using 4 μL of extracted total RNA as a template, add MgCl 2 3 μL, 10 × reverse transcription buffer 2.5 μL, dNTP Mixture (10 mmol/L) 2.5 μL, RNaseInhititor 0.5 μL (20U), RT enzyme 0.5 μL, Taq Enzyme 0.5 μL, primer P1 1 μL, primer P2 1 μL, sterilized dH 2 O9.5 μL, total system 25 μL. Perform the RT-PCR reaction under the following reaction conditions: 50 ℃ 40 s, 94 ℃ 2 min, 94 ℃ 45 s, 53 ℃ 90 s, 72 ℃ 100 s, cycle 30 times, and finally 72 ℃ extension 10 min. After the PCR product was identified by electrophoresis on a 1% agarose gel containing ethidium bromide, the target band was cut, and then recovered according to the gel recovery kit instructions to be the Newcastle disease virus HN gene.
[0073] (2) VP2 33-221aa Acquisition of gene sequence
[0074] According to the IBDV VP2 nucleotide sequence in GenBank (No:AF508177), a pair of specific primers P3 and P4 were designed to amplify VP2 using DNA Star7.0 and Primerpremier6.0 software 33-221aa. The primers were synthesized by Dalian Bao Biological Engineering Company. Primer P3 5’ end is introduced into the flexible joint, P4 5’ end Hin dⅢ endonuclease site. The primer sequence is as follows:
[0075] Table 2 The primer sequences used in PCR amplification
[0076]
[0077] The pMD18-T-VP2 constructed in the previous stage was used as a template to amplify chicken VP2 by PCR 33-221aa. Add 10×PCR Buffer, 5 μL, MgCl to the reaction system 2 , 3 μL; dNTP, 2.5 mmol/L, 4 μL; pMD18-T-VP2 template 0.5 μL, primer P3 and primer P4, the final concentration is 10 pmol/L each 2 μL; TaKaRa Ex Taq 0.5 μL; sterilized ultrapure water, 33 μL; reaction conditions: 95 ℃ pre-denaturation 5 min; 95 ℃ denaturation 30 s; 63 ℃ annealing for 30 s; 72 ℃ extension 40 s, 30 cycles; 72 ℃ extension 10 min . After the PCR reaction is over, perform agarose gel electrophoresis and observe the results. After the PCR amplification product was identified by 1% agarose gel electrophoresis, the target band was cut out and recovered and purified using the ordinary agarose gel DNA recovery kit of Dalian TaKaRa Company. The purified product is VP2 33-221aa The gene fragments will be sent to Dalian Biotech for sequencing.
[0078] (3) HN-VP2 33-221aa Acquisition of fusion protein coding gene sequence
[0079] Use the above recovered and purified HN/VP2 gene fragment as a template, use P1/P4 primers, and obtain HN-VP2 by SOEing PCR technology 33-221aa Fusion fragments.
[0080] PCR reaction system 50 μL: 10×PCR Buffer, 5 μL, MgCl 2 , 3 μL; dNTP, 10 mmol/L, 1 μL; VP2 33-221aa And Newcastle disease virus HN gene as templates, add 5 μL each, primer P1 and primer P4, the final concentration is 20 pmol/L each 2 μL; TaKaRa Ex Taq 0.5 μL; sterilized ultrapure water, 34.5 μL;
[0081] PCR reaction conditions: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s; 63 ℃ annealing for 30 s; 72 ℃ extension for 2 min 20 s, 30 cycles; 72 ℃ extension for 10 min, PCR product containing 1% ethidium bromide After identification by agarose gel electrophoresis, the target band is cut out, and then recovered according to the instructions of the gel recovery kit, which is the HN-VP2 fusion protein encoding gene (SEQ.ID.NO.5).
[0082] 2. Recombinant plasmid pET32a-HN-VP2 33-221aa Construction and identification of
[0083] First, HN-VP2 33-221aa Link to pMD19-T vector, and then use Bam HⅠ, Hin dⅢ to pMD19-T-HN-VP2 33-221aa Carry out double enzyme digestion and recover the fusion gene fragment HN-VP2 33-221aa. At the same time use pET32a Bam HⅠ, Hin dⅢ Perform double digestion and recover the vector fragment. The recovered HN-VP2 33-221aa Connect with pET32a, ligation system 10 µL: target fragment 6 µL, vector 2 µL, T4 DNA ligase 1 µL, Buffer 1 µL. The connection conditions are 12-16°C overnight. Mix the Rosetta competent cells and the ligation product in a test tube in an ice bath for 30 min, heat shock at 42°C for 90 s, and ice bath for 2 min. Add 400 µL of LB medium to the test tube, and incubate in a constant temperature shaker at 37°C for 1.5 h , Take 100 µL for plating (LB+AMP), incubate at 37°C for 10-12 hours, pick a single colony for PCR and double enzyme digestion identification, such as Figure 1-2 , The plasmid with a vector band of about 5900bp and a DNA fragment of 2300bp after digestion was used as the positive plasmid, and the positive plasmid was named pET32a-HN-VP2 33-221aa , Sent to Shanghai Invitrongen company for sequencing.
[0084] Three, express HN-VP2 33-221aa Construction of genetically engineered strains of fusion protein:
[0085] Take the positive recombinant expression vector pET32a-HN-VP2 obtained above 33-221aa Mix 5 μg with 80 μl LRosetta competent cells, mix well in a test tube and ice bath for 30 min, then heat shock at 42°C for 90 s, then ice bath for 2 min, finally add 400 μL LB medium to the test tube, culture for 1.5 h, then Coat the plate with 100 µL (LB+AMP), culture for 10~12 h, pick a single colony for PCR identification, the reaction system is the same as above, PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 63℃ annealing for 30 s; 72 ℃ extension for 2 min 20 s, 30 cycles; 72 ℃ extension for 10 min, amplify a 2300bp gene fragment, and transfer the identified single colony to LB+AMP liquid medium, extract the positive plasmid, and then use Bam HⅠ, Hin dⅢ Double enzyme digestion identification, the digestion system is 10×H buffer 1 µL, Bam HⅠ 0.4 µL, Hin dⅢ 0.4 µL, recombinant plasmid 8.2 µL; digestion conditions are all 37ºC constant temperature water bath for 3 hours. PCR and restriction enzyme digestion identify the correct colony as expressing HN-VP2 33-221aa A genetically engineered E. coli strain for the fusion protein.
[0086] Fourth, reorganize HN-VP2 33-221aa Induced expression and identification of fusion protein:
[0087] Will identify the correct Rosetta (pET32a-HN-VP2 33-221aa ) A single colony is transferred to the LB+AMP liquid medium and cultured for 10-12 hours, and the cultured bacterial liquid and medium are expanded and cultured for about 5 hours after 1:100. Add 80 µL of inducer IPTG (the ratio of inducer to bacterial solution is 1:50) and shake for 6 h. After that, take out the bacterial solution, put the induced bacteria into a 1.5 mL EP tube, centrifuge, remove the supernatant, add PBS or 200 µL of ultrapure water to wash 2~3 times, and finally add 200 µL of ultrapure water to disrupt the cells with ultrasound The instrument is broken until the solution is transparent. Then add 50 µL of 5× sample solution (the ratio of sample solution to solution is 1:4) into the solution, boil it in boiling water for 10 minutes, and mark it.
[0088] Prepare 5 mL separation gel, use a 5 mL pipette to take an appropriate amount into the gel plate, add an appropriate amount of isobutanol to eliminate bubbles, and let it stand for about 1 h until the separation gel is solidified; use filter paper to completely suck out the isobutanol and prepare 2 mL concentrate Add the gel to the gel plate, quickly insert the comb, and let it stand for 1 hour for the gel to solidify; place the gel plate in the appropriate position in the electrophoresis tank, add appropriate buffer, pull out the comb for spotting, connect the power supply, and concentrate the gel with constant pressure Run at 80 V for about 45 minutes, wait until the bromophenol blue runs through the concentrated gel, and then keep the pressure at 120 V until some of the bromophenol blue runs out of the buffer in the sample, and disconnect the power supply.
[0089] Take out the electrophoresis gel, stain it with Coomassie Brilliant Blue for 1 h, then decolorize it with a decolorizing solution, change it every 2 h, and repeat about 4 times. Analyze protein expression in a gel imager. The recombinant HN-VP233-221aa fusion protein has a molecular weight of about 104 kDa, such as image 3.
[0090] At the same time, the NDV positive serum was used as the primary antibody for Western blotting. The lane where the recombinant E. coli culture solution was loaded had a specific band of interest, while the lane where the negative control was loaded did not appear. Figure 4. It shows that the recombinant HN-VP233-221aa fusion protein has been successfully expressed and has good immunoreactivity.
