Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (8): 1620-1628.doi: 10.3864/j.issn.0578-1752.2024.08.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Preparation and Application of Polyclonal Antibodies Against Pig CD1d Protein

LIU ChuanXia(), CHEN Xin, WANG Xiao, LI XueWen, LI TingTing, WENG ChangJiang, ZHENG Jun()   

  1. Division of Fundamental Immunology, National African Swine Fever Para-Reference Laboratory, State Key Laboratory for Animal Disease Control and Prevention/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069
  • Received:2022-11-17 Accepted:2024-03-01 Online:2024-04-16 Published:2024-04-24
  • Contact: ZHENG Jun

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Abstract:

【Objective】 The aim of this study was to prepare polyclonal antibodies against porcine CD1d protein, so as to lay the foundation for exploring the function of porcine CD1d protein in the process of African swine fever virus (ASFV) infection. 【Method】 In this study, the pig CD1d gene was amplified using PCR and homologously recombined into the pGEX-6p1 vector, constructing a prokaryotic recombinant expression plasmid pGEX-6p1-CD1d. The recombinant plasmid E. coli BL21 (DE3) was transformed and induced for expression using IPTG. The expressed GST CD1d recombinant protein was identified by SDS-PAGE and Western blot (WB) methods. The SDS-PAGE results showed a clear band at approximately 50 ku, which was expressed in the form of an inclusion body. Then, protein purification was performed using glutathione agarose affinity chromatography. The purified GST-CD1d protein was mixed and emulsified with an equal volume of Freund's complete adjuvant. The purified protein was immunized in New Zealand white rabbits and administered subcutaneously at multiple points on the neck and back, with an immune dose of 200 μG/piece, and then second and third immunizations were performed at the 3rd and 5th weeks after the first immunization, respectively, using Freund's incomplete adjuvant emulsification, with the same method and dosage as the first immunization. On the 7th day after the third immunization, the blood was collected from the ear vein to isolate the serum. The fourth immunization was conducted at the 7 weeks after the first immunization, and the blood was collected from the heart one week later. The antibody was purified by Protein G affinity chromatography and frozen at -80 ℃. The expression and cellular localization of endogenous CD1d protein expressed by transient transfection of exogenous and porcine primary macrophages (PAMs) were indentified by using WB and indirect immunofluorescence (IFA). Similarly, the prepared CD1d antibody could pull down CD1d expressed by transient exogenous transfection through IP. In order to investigate the early stage of ASFV infection, ASFV was inoculated into PAMs and samples of ASFV infection for 0, 15, 30, and 60 minutes were prepared, respectively. CD1d was used as the primary antibody and the expression of CD1d protein was detected by WB. Plasmids pCAGGS-HA-CD1d and pCAGGS-Flag-CD2v were co transfected into HEK293T cells. After 24 hours, the cells were collected for lysis, and Flag beads overnight binding protein was added. The interaction was detected by WB staining. At the same time, the plasmids were cotransfected into HEK293T cells in a confocal dish, incubated with labeled antibodies, and corresponding fluorescent secondary antibodies were selected. The co localization of CD1d and CD2v was observed under a laser confocal microscope. Verification of Co-IP interaction between CD1d and ASFV outer capsule protein CD2v was verified. 【Result】 The GST-CD1d protein expressed in prokaryotic cells was expressed in the form of inclusion bodies, with a molecular weight of approximately 35 ku; After four rounds of immunization with CD1d recombinant protein in experimental rabbits, blood was collected and serum was separated. The purified antibody was detected by SDS-PAGE and showed a specific band at 45 and 25 ku, respectively, representing the heavy and light chains of the CD1d antibody. The rabbit anti CD1d antibody prepared using purified CD1d protein as immunogen contained both heavy and light chains, and had good purity; This antibody could identify the expression and cellular localization of transient transfected exogenous and PAMs endogenous CD1d proteins through WB and IFA. Further testing results showed that after ASFV infection with PAMs, the expression level of CD1d protein significantly increased, and WB and IFA results showed that CD1d interacted and co localized with the outer capsule protein CD2v encoded by ASFV. 【Conclusion】 This study prepared antibodies against CD1d through prokaryotic expression technology, laying the foundation for further exploration of the biological function of CD1d protein in ASFV infection process.

Key words: CD1d protein, prokaryotic expression, polyclonal antibody, African swine fever virus, CD2v protein

Fig. 1

Identification of porcine CD1d protein expression and purification A: Identification by SDS-PAGE; B: Identification of WB. M: Protein molecular quality standard; 1: The bacterial liquid without induction; 2-4: The recombinant bacterial liquid induced by IPTG, precipitation after ultrasound, supernatant after ultrasound; 5: Purified CD1d protein was identified by WB; 6: Purified CD1d protein was identified by SDS-PAGE"

Fig. 2

SDS-PAGE identification of CD1d antibody M: Protein molecular quality standard; 1-4: Purified CD1d antibodies in different concentrations"

Fig. 3

Identification of the expression and localization of exogenous CD1d protein A: The expression of exogenous CD1d protein in HEK293T cells was analyzed by WB; B: The localization of exogenous CD1d protein in HEK293T cells was identified by IFA. M: Protein molecular quality standard; 1: lysate of HEK293T cells transfected with pCAGGS-HA-CD1d; 2: Lysate of HEK293T cells transfected with pCAGGS-HA"

Fig. 4

Identification of endogenous CD1d protein expression and localization A: The expression of CD1d in PAMs was analyzed by WB. B: Identification of CD1d localization in PAMs by IFA. M: Protein molecular quality standard; 1: lysate of PAMs infected with ASFV; 2: Uninfected PAMs lysate"

Fig. 5

Detection of CD1d antibody by IP test"

Fig. 6

Effect of ASFV infection on CD1d protein expression level A: The expression of CD1d at different time of ASFV infection was analyzed by WB; B: Compare and analyze the gray value of Figure A. The ordinate value shows the gray ratio of CD1d and GAPDH"

Fig. 7

CD1d interacts with the envelope protein CD2v of African swine fever virus A: The colocalization of CD1d and CD2v was identified by IFA; B: The interaction between CD1d and CD2v was identified by Co-IP"

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