Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (8): 142-149.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1404
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QIN Xue-jing(), WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun()
Received:
2021-11-10
Online:
2022-08-26
Published:
2022-09-14
Contact:
ZHANG Ling-yun
E-mail:Krystal529142879@163.com;lyzhang@bjfu.edu.cn
QIN Xue-jing, WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun. Prokaryotic Expression and Preparation of Polyclonal Antibody of PwHAP5 Gene in Picea wilsonii[J]. Biotechnology Bulletin, 2022, 38(8): 142-149.
引物名称 Primer name | 序列 Sequence(5'-3') | 酶切位点 Enzyme |
---|---|---|
上游引物P1 | CGCGGATCCTATGGATCAGCAGCAGC-CCACAAT | BamH I |
下游引物P2 | CCCAAGCTTACTGCTGCCATGAGGA- GGTG | Hind III |
Table 1 Quote sequence information
引物名称 Primer name | 序列 Sequence(5'-3') | 酶切位点 Enzyme |
---|---|---|
上游引物P1 | CGCGGATCCTATGGATCAGCAGCAGC-CCACAAT | BamH I |
下游引物P2 | CCCAAGCTTACTGCTGCCATGAGGA- GGTG | Hind III |
Fig. 1 PCR products of PwHAP5 and identification of the recombinant plasmid A:PCR products of PwHAP5(M:DL2000; 1:RT-PCR). B:Identification of the plasmid pET-48b-PwHAP5(M:DL2000; 1:the plasmid pET-48b-PwHAP5 digested with BamH I and Hind Ⅲ)
Fig.2 Identification of expression form and expression amount of target protein PwHAP5 A:Identification on the expression form of target protein(1:Recombinant E. coli pET-48b-PwHAP5/BL2;2:the lysate supernatant of E. coli pET-48b-PwHAP5/BL2;3:the lysate precipitant of E. coli pET-48b-PwHAP5/BL2);B:SDS-PAGE of the target protein(1:the target protein without IPTG induction;2-4:the target protein induced for 4,6 and 8 h after IPTG,respectively)
Fig.3 SDS-PAGE analysis of purified recombinant protein and Western blotting assay A:Purified recombinant protein via Ni-NTA(1:protein marker;2:the total protein;3-5:the unnecessary proteins in the 1st,2nd and 3rd eluents,respectively;6-8:the necessary proteins in the 1st,2nd and 3rd eluents,respectively). B:Western blotting assay(a and b:serum from 2 different rabbits;1:protein marker;2:the rabbit antiserum)
Fig.7 Pollen tube germination test and indirect immuno-fluorescence assay A:Germination situation of pollen tube under different time treatment;B:indirect immunofluorescence detection of antibody(a, d:the control group;b, e:the primary antibody treated pollen tube;e, f:pollen tubes treated with primary and secondary antibodies)
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