Prokaryotic Expression and Preparation of Polyclonal Antibody of PwHAP5 Gene in Picea wilsonii

doi:10.13560/j.cnki.biotech.bull.1985.2021-1404

Abstract

Abstract:

Specific polyclonal antibodies against PwHAP5 gene of Picea wilsonii were prepared to provide the experimental data for follow-up studies of PwHAP5 protein localization,expression and function. The PwHAP5 gene fragment from P. wilsonii pollen was amplified by PCR and then was inserted into vector pET-48b for constructing the recombinant plasmid pET-48b-PwHAP5. The recombinant protein of PwHAP5 was induced and was purified by His-NTA nickel column. The expressed recombinant protein was tested by SDS-PAGE and injected into New Zealand rabbits as an antigen to obtain antiserum. Antiserum was purified by ProteinA,the purity and specificity of the antibody was identified by SDS-PAGE and Western blot method. As results,the prokaryotic expression vector pET-48b-PwHAP5 was successfully constructed,and the PwHAP5 protein with molecular weight of 45 kD was prepared. The titer of PwHAP5 rabbit antiserum was more than 1∶729 000 via indirect ELISA. The prepared PwHAP5 protein of rabbit polyclonal antibody has a high titer and specifically recognize antigen,which can meet the requirements of subsequent studies on the functions of PwHAP5 in the growth and development of P. wilsonii.

Key words: PwHAP5 gene, prokaryotic expression, protein purification, polyclonal antibody

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.

Figures/Tables 8

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.4 Detected titer of antibody detected by indirect ELISA

Fig.5 Purity detection of purified antibody 1：Protein marker;2：the antibody

Fig. 6 Specificity of rabbit polyclonal antibody of PwHAP5 1：Protein of PwHAP5;2：uninduced protein of PwHAP5;3：the control group

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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