Polyclonal Antibody Preparation Research Articles

Preparation of polyclonal antibodies to chicken P62 protein and its application in nephropathogenic infectious bronchitis virus-infected chickens

Preparation of polyclonal antibodies to chicken P62 protein and its application in nephropathogenic infectious bronchitis virus-infected chickens

Preparation of polyclonal antibodies to the chicken Beclin1 protein and its application in the detection of nephropathogenic infectious bronchitis virus

Beclin1, also known as ATG6, has been shown to be closely related to coronavirus, however, the link between Beclin1 and nephropathogenic infectious bronchitis virus (NIBV) has been poorly investigated and there are no available antibodies specifically targeting the chicken Beclin1 protein. The study aimed to prepare and assay a polyclonal antibody to Beclin1, enabling a deeper understanding of the mechanism of action of Beclin1 in NIBV. In this study, we amplified the chicken Beclin1 target gene and constructed a recombinant plasmid using prokaryotic expression techniques, then obtained the recombinant target protein by induced expression. Finally, the serum is obtained by immunizing rabbits with the purified and concentrated protein. The results show that the antiserum potency of the ELISA assay was >1:204800. By western blotting and immunofluorescence, the antibodies we prepared specifically recognized the chicken Beclin1 protein, which is mainly found in the nucleus of trachea, lung, kidney, spleen and fabricant cells. NIBV infection significantly decreased the expression of Beclin1 in the trachea, but increased in others. We have successfully prepared specific rabbit anti-chicken Beclin1 polyclonal antibodies, and detected changes in tissues of diseased chickens infected with NIBV, laying the foundation for further studies on the role of Beclin1 in avian diseases.

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis

In order to investigate the crucial role of ZmBT1 in starch accumulation during maize grain development and analyze the expression and distribution of ZmBT1 in various maize tissues, we prepared a polyclonal antibody. Specifically, we successfully expressed the recombinant plasmid pGEX-6p-ZmBT1-C (382-437aa) and purified Gst-ZmBT1-C as the antigen for antibody preparation. Our results confirmed that the ZmBT1 protein in maize tissues can be specifically recognized by the ZmBT1 antibody. Through Western blotting, we observed that the expression protein of ZmBT1 varied by tissues, with the highest content in the grain and endosperm. Furthermore, we employed a combination of Western blotting and quantitative real-time PCR to show that the expression level of ZmBT1 can be influenced by plant hormones. This finding suggests that ZmBT1 plays a critical role in the accumulation of starch and opens up new avenues for functional studies of this protein.

Antibody-mediated antigen loss switches augmented immunity to antibody-mediated immunosuppression

AbstractAntibodies against fetal red blood cell (RBC) antigens can cause hemolytic disease of the fetus and newborn (HDFN). Reductions in HDFN due to anti-RhD antibodies have been achieved through use of Rh immune globulin (RhIg), a polyclonal antibody preparation that causes antibody-mediated immunosuppression (AMIS), thereby preventing maternal immune responses against fetal RBCs. Despite the success of RhIg, it is only effective against 1 alloantigen. The lack of similar interventions that mitigate immune responses toward other RBC alloantigens reflects an incomplete understanding of AMIS mechanisms. AMIS has been previously attributed to rapid antibody-mediated RBC removal, resulting in B-cell ignorance of the RBC alloantigen. However, our data demonstrate that antibody-mediated RBC removal can enhance de novo alloimmunization. In contrast, inclusion of antibodies that possess the ability to rapidly remove the target antigen in the absence of detectable RBC clearance can convert an augmented antibody response to AMIS. These results suggest that the ability of antibodies to remove target antigens from the RBC surface can trigger AMIS in situations in which enhanced immunity may otherwise occur. In doing so, these results hold promise in identifying key antibody characteristics that can drive AMIS, thereby facilitating the design of AMIS approaches toward other RBC antigens to eliminate all forms of HDFN.

Preparation of polyclonal antibody against a universal bacterial antigen OmpA deduced by bioinformatic analysis and preliminary evaluation of concentration effects on foodborne pathogens

Rapid and ultrasensitive microbial detection in actual samples have challenges because of target pathogen diversity and low abundance. In this study, we attempted to capture and concentrate multiple pathogens by combining magnetic beads with polyclonal antibodies against a universal antigen of ompA, LAMOA-1, before further detection. A protein sequence consisting of 241 amino acids with spatial conformation similar to E. coli ompA was identified and expressed as a recombinant protein in prokaryotes according to the results of sequence alignment among 432 sequences of ompA belonging to intestinal bacteria from gram-negative bacteria. Purified from immunized rabbits, the anti-LAMOA-1 antibody was shown to effectively recognize 12 foodborne bacterial species. Antibody-conjugated beads were used to concentrate the bacteria when the bacterial concentration in artificially contaminated samples is between 10 and 100 CFU/mL, which shortens detection duration by 8–24 h. The enrichment strategy is potentially beneficial for detection of foodborne pathogens.

Rapid detection of apple latent spherical virus infecting Angelica sinensis by gold immunochromatography reverse transcription loop-mediated isothermal amplification assay

Apple latent spherical virus (ALSV; genus: Cheravirus, family: Secoviridae) is capable of infecting Angelica sinensis under natural conditions and causing symptoms of greenish discoloration and mottling, affecting plant growth and causing a reduction in root quality and yield. Virus detection of ALSV is an urgent need for the continued development of angelica industry. Consequently, it is necessary to develop simple, sensitive, and reliable ALSV detection methods. We describe the preparation of polyclonal antibodies with high titer and high specificity using recombinant ALSV coat protein (CP) as the antigen. Using these antibodies, we established and optimized a rapid detection system using colloidal gold immunochromatography assay (GICA) to detect ALSV. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system with a minimum detection line of 2.72 × 103copys/μl was also developed to detect ALSV. This assay was further enhanced using a combination GICA-RT-LAMP assay. This GICA assay successfully detected ALSV in infected plants without cross reactivity recorded from three other plant viruses. The sensitivity of GICA was the same as that of GICA-RT-PCR, whereas the sensitivity of GICA-RT-LAMP was at least 10-fold higher than that of GICA and GICA-RT-PCR. These methods can detect and identify ALSV under field conditions and accurately measure virus concentration in the laboratory.

Ruminal fermentation pattern of acidosis-induced cows fed either monensin or polyclonal antibodies preparation against several ruminal bacteria.

This study was designed to evaluate a spray-dried multivalent polyclonal antibody preparation (PAP) against lactate-producing bacteria as an alternative to monensin (MON) to control ruminal acidification. Holstein cows (677 ± 98 kg) fitted with ruminal cannulas were allocated in an incomplete Latin square design with two 20 days period. Cows were randomly assigned to control (CTL), PAP, or MON treatments. For each period, cows were fed a forage diet in the first 5 days (d-5 to d-1), composed of sugarcane, urea and a mineral supplement, followed by a 74% concentrate diet for 15 days (d 0 to d 14). There were no treatment main effects (P > 0.05) on dry matter intake (DMI) and microbial protein synthesis. However, there was a large peak (P < 0.01) of intake on d 0 (18.29 kg), followed by a large decline on d 1 (3.67 kg). From d2, DMI showed an increasing pattern (8.34 kg) and stabilized around d 8 (12.96 kg). Higher mean pH was measured (P < 0.01) in cattle-fed MON (6.06 vs. PAP = 5.89 and CTL = 5.91). The ruminal NH3-N concentration of CTL-fed cows was lower (P < 0.01) compared to those fed MON or PAP. The molar concentration of acetate and lactate was not affected (P > 0.23) by treatments, but feeding MON increased (P = 0.01) propionate during the first 4 days after the challenge. Feeding MON and PAP reduced (P = 0.01) the molar proportion of butyrate. MON was effective in controlling pH and improved ruminal fermentation of acidosis-induced cows. However, PAP was not effective in controlling acidosis. The acidosis induced by the challenge was caused by the accumulation of SCFAs. Therefore, the real conditions for evaluation of this feed additive were not reached in this experiment, since this PAP was proposed to work against lactate-producing bacteria.

SARS-CoV-2 variants resistant to monoclonal antibodies in immunocompromised patients constitute a public health concern.

COVID-19 in immunocompromised hosts has emerged as a difficult therapeutic management problem. Immunocompromised hosts mount weak responses to SARS-CoV-2 and manifest infection outcomes ranging from severe disease to persistent infection. Weakened immune systems mean greater viral loads and increased opportunities for viral evolution. Gupta, Konnova, et al. report the emergence of resistant SARS-CoV-2 variants in immunocompromised patients after monoclonal antibody (mAb) therapy. mAbs target only a single determinant in the viral Spike protein, which is a weakness of such therapy when treating a mutagenic and variable virus. Hence, the emergence of mAb resistance could have been anticipated, but its documentation is important because it has major public health implications, since such resistant variants have the potential to spread and escape vaccine immunity. For immunocompromised patients, these findings suggest the need for combination therapy with antiviral drugs and the use of polyclonal antibody preparations such as convalescent plasma.

Preparation of polyclonal anti-Schistosoma mansoni cysteine protease antibodies for early diagnosis

In many parts of the tropics, schistosomiasis is a major parasitic disease second only to malaria as a cause of morbidity and mortality. Diagnostic approaches include microscopic sampling of excreta such as the Kato-Katz method, radiography, and serology. Due to their vital role in many stages of the parasitic life cycle, proteases have been under investigation as targets of immunological or chemotherapeutic anti-Schistosoma agents. Five major classes of protease have been identified on the basis of the peptide hydrolysis mechanism: serine, cysteine, aspartic, threonine, and metalloproteases. Proteases of all five catalytic classes have been identified from S. mansoni through proteomic or genetic analysis. The study aimed to produce polyclonal antibodies (pAbs) against schistosomal cysteine proteases (CP) to be used in the diagnosis of schistosomiasis. This study was conducted on S. mansoni-infected patients from highly endemic areas and from outpatients’ clinic and hospitals and other patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids). In this study, the produced polyclonal antibodies against S. mansoni cysteine protease antigens were labeled with horseradish peroxidase (HRP) conjugate and used to detect CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA. The study involved 200 S. mansoni-infected patients (diagnosed by finding characteristic eggs in the collected stool samples), 100 patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids), and 100 individuals who served as parasite-free healthy negative control. The prepared pAb succeeded in detecting CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA with a sensitivity of 98.5% and 98.0% respectively. A positive correlation was observed between S. mansoni egg counts and both stool and serum antigen concentrations. Purified 27.5 kDa CP could be introduced as a suitable candidate antigen for early immunodiagnosis using sandwich ELISA for antigen detection.Key points• Detection of cysteine protease antigens can replace parasitological examination.• Sandwich ELISA has a higher sensitivity than microscopic examination of eggs.• Identification of antigens is important for the goal of obtaining diagnostic tools.

A peptide derived from the N-terminal of NS2A for the preparation of ZIKV NS2A recognition polyclonal antibody

Zika virus non-structural protein NS2A participates in viral replication, organization, and budding, as well as escaping host immunity. NS2A also involved in the induction of microcephaly by ZIKV. However, the above studies were mainly performed through NS2A with a tag due to the lack of available antibodies against NS2A. ZIKV NS2A is a multiplex transmembrane protein, which leads to difficulties in the preparation of its recognition antibodies, thus seriously affecting the study of ZIKV NS2A. In this study, we found that a peptide (GSTDHMDHFSLGVLC) derived from the N-terminal of ZIKV NS2A coupled to KLH induced antibodies recognizing ZIKV NS2A in rabbits. The purified polyclonal antibody recognized ZIKV NS2A in ZIKV-infected cells with high efficiency and specificity, as detected by western blot and immunofluorescence assay. Our study has important implications for the preparation of ZIKV NS2A antibodies and the in-depth study of ZIKV NS2A.

Preparation, Purification and Performance Evaluation of Polyclonal Antibody Against SARS-CoV-2 Produced in Rat.

Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats. Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software. The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification. Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

Preparation of the RIPK3 Polyclonal Antibody and Its Application in Immunoassays of Nephropathogenic Infectious Bronchitis Virus-Infected Chickens.

Receptor interacting protein kinase 3 (RIPK3) is a vital serine/threonine kinase in regulating the programmed destruction of infected cells to defend against RNA viruses. Although the role of RIPK3 in viruses in mice is well characterized, it remains unclear where in nephropathogenic infectious bronchitis virus (NIBV) in chickens. Here, we use a self-prepared polyclonal antibody to clarify the abundance of RIPK3 in tissues and define the contributions of RIPK3 in tissue damage caused by NIBV infection in chickens. Western blot analyses showed that RIPK3 polyclonal antibody can specifically recognize RIPK3 in the vital tissues of Hy-Line brown chicks and RIPK3 protein is abundantly expressed in the liver and kidney. Moreover, NIBV significantly upregulated the expression levels of RIPK3 in the trachea and kidney of chicks in a time-dependent manner. In addition, the activation of necroptosis in response to NIBV infection was demonstrated by the coimmunoprecipitation (CoIP) experiments through RIPK3 in the necrosome, which phosphorylates its downstream mixed-spectrum kinase structural domain-like protein (MLKL). Our findings offered preliminary insights into the key role of RIPK3 protein in studying the underlying mechanism of organ failure caused by NIBV infection.

Research Note: Transcriptomic analysis of LMH cells in response to the overexpression of a hypothetical protein identified in Eimeria tenella SD-01 strain

Though genome sequencing of Eimeria tenella predicts more than 8,000 genes, the molecular functions of many proteins remain unknown. In this study, the coding region corresponding to the mature peptide of a hypothetical protein of E. tenella (ETH_00023950) was amplified and expressed in a bacterial system. Following preparation of polyclonal antibody that recognizes ETH_00023950, the expression of ETH_00023950 in merozoites was examined. Meanwhile, we determined the transcriptomic responses of the leghorn male hepatoma (LMH) cells to its expression. Sequencing analysis showed that one single nucleotide polymorphism and one indel of ETH_00023950 of E. tenella SD-01 strain were found compared with that of the UK reference Houghton strain, leading to a frame shift and a premature stop codon. The expression of ETH_00023950 in E. tenella merozoites was confirmed by indirect immunofluorescence and Western blot analysis. Transcriptomic analysis showed that ETH_00023950 altered the expression of 2,680 genes (321 downregulated genes and 2,359 upregulated genes) in LMH cells. The RNA-sequencing data were consistent with the results of the quantitative real-time polymerase chain reaction (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that differentially expressed transcripts were significantly related to 8 pathways, including oxidative phosphorylation and TGF-beta signaling pathway. These findings contribute to understanding host-pathogen interaction and secondary bacterial infections related to E. tenella.

Preparation of polyclonal antibody against phosphatidylethanolamine binding protein 1 recombinant protein and its functional verification in pulmonary hypertension syndrome in broilers

Pulmonary hypertension syndrome (PHS) is a disease that is difficult to overcome for fast-growing broilers. It causes pulmonary vascular remodeling and ascites in broilers. As a classical inhibitor of cancer metastasis, phosphatidylethanolamine binding protein 1 (PEBP1) regulates angiogenesis in the process of tumor metastasis through multiple signal pathways. However, whether PEBP1 can regulate pulmonary artery remodeling in broilers with PHS has not been reported. This study constructed the prokaryotic expression vector of [PEBP1]-pET32a by genetic engineering technology, the recombinant PEBP1 protein was expressed in large quantities, and the PEBP1 polyclonal antibody was prepared by immunizing rabbits with the recombinant PEBP1 protein. Western blot and immunofluorescence results showed that PEBP1 was expressed in many kinds of animal tissues. However, due to the species specificity of polyclonal antibodies, the expression level of PEBP1 protein in broilers and ducks with high homology was significantly higher than that in other species of animals. More interestingly, we found that the expression of PEBP1 protein decreased significantly in broilers with PHS. These studies laid a foundation for further exploration of the mechanism of pulmonary artery remodeling. In addition, the PEBP1 polyclonal antibody provided convenience for further study of the role of PEBP1 in PHS.

SARS-CoV-2 BA.1 variant is neutralized by vaccine booster-elicited serum but evades most convalescent serum and therapeutic antibodies.

The rapid spread of the highly contagious Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with its high number of mutations in the spike gene has raised alarms about the effectiveness of current medical countermeasures. To address this concern, we measured neutralization of the Omicron BA.1 variant pseudovirus by post-vaccination serum samples after two and three immunizations with the Pfizer/BioNTech162b2 SARS-CoV-2 mRNA (Pfizer/BNT162b2) vaccine, convalescent serum samples from unvaccinated individuals infected by different variants, and clinical-stage therapeutic antibodies. We found that titers against the Omicron variant were low or undetectable after two immunizations and in many convalescent serum samples, regardless of the infecting variant. A booster vaccination increased titers more than 30-fold against Omicron to values comparable to those seen against the D614G variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. We also evaluated eighteen clinical-stage therapeutic antibody products and an antibody mimetic protein product obtained directly from the manufacturers. Five monoclonal antibodies, the antibody mimetic protein, three antibody cocktails, and two polyclonal antibody preparations retained measurable neutralization activity against Omicron with a varying degree of potency. Of these, only three retained potencies comparable to the D614G variant. Two therapeutic antibody cocktails in the tested panel that are authorized for emergency use in the United States did not neutralize Omicron. These findings underscore the potential benefit of mRNA vaccine boosters for protection against Omicron and the need for rapid development of antibody therapeutics that maintain potency against emerging variants.

Polyclonal antibody preparations from avian origin as a feed additive to beef cattle: ruminal fermentation during the step-up transition diets.

This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40%, 35%, and 25% of the preparation, respectively) on ruminal fermentation [pH, ammonia-N (NH3-N), lactate, and volatile fatty acids (VFA)] of beef steers during a 21-d step-up diet adaptation. Eight ruminally cannulated Angus crossbred beef steers (658 ± 79 kg of body weight) were assigned in a crossover design to be transitioned from a diet containing ad libitum bermudagrass hay [Cynodon dactylon (L.) Pers.] plus 0.45 kg/d (as fed) of molasses with 0 (CON) or 3 g of PAP (PAP) to a high-grain diet. Transition consisted of three 7-d steps of increased inclusion of cracked corn (35%, 60%, and 82% of the diet DM for STEP1, STEP2, and STEP3, respectively). On each transition day and 7 d after STEP3 (STEP3-7d), ruminal fluid samples were obtained every 3 h for 24 h. Feeding 3 g of PAP daily increased (P < 0.01) average ruminal pH during STEP3 compared with CON steers (5.6 vs. 5.4 ± 0.05, respectively). During STEP1, NH3-N concentration was greater (P < 0.01; 9.4 vs. 6.8 ± 0.74 mM, respectively), and time (min/d) and area (time × pH) of ruminal pH below or equal to 5.2 was lesser (P ≤ 0.03) for steers consuming PAP compared with steers assigned to CON treatment (33.4 vs. 73.3 ± 21.7 min/d and 187.4 vs. 406.3 ± 119.7 min × pH/d, respectively). Steers consuming PAP had greater acetate:propionate ratio at 0, 3, and 6 h relative to diet change compared with CON (2.42, 2.35, 2.29 vs. 1.66, 1.79, and 1.72 ± 0.17, respectively), whereas butyrate molar proportions increased (P = 0.02; 17.1 vs. 11 ± 1.58 mol/100 mol for CON and PAP, respectively) when PAP was not fed at STEP2. Total ruminal lactate concentrations were not affected by PAP feeding (P > 0.11). In conclusion, feeding 3 g/d of polyclonal antibody preparation against S. bovis, F. necrophorum, and lipopolysaccharides was effective in increasing ruminal pH, A:P ratio, and NH3-N concentrations, possibly attenuating the risks of ruminal acidosis in steers during the step-up transition from forage to high-grain diets.

Preparation of polyclonal antibodies against chemically synthesized ApxIA and ApxIVA toxins and their diagnostic efficacy in the experimentally injected mice

ObjectivesApxIA and ApxIVA are the main virulence factors of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). The preparation of their antibodies is of great significance for diagnosing and treating porcine pleuropneumonia. MethodsThis study used chemically synthesized ApxIA and ApxIVA peptides as immunogens to prepare polyclonal antibodies against ApxIA and ApxIVA. Their titers were determined by ELISA and Dot blotting. Histopathology of apparently infected organs was carried out. Finally, the immunofluorescence method detected the distribution of antigens in the visceral tissues of mice infected with ApxIA and ApxIVA toxins. ResultsThe results showed that ApxIA and ApxIVA polyclonal antibodies with titer as high as 1: 200000 and 1: 50000, respectively, were successfully obtained. The immunofluorescence results showed that these polyclonal antibodies could accurately detect ApxIA and ApxIVA antigens in mouse lungs with strong specificity. Histopathologically, there were perialveolar hemorrhages, pulmonary emphysema, and sloughing of bronchiolar epithelium. There was vacuolation and infiltration of chronic inflammatory cells in the liver parenchymal tissue. Atrophy of intestinal villi and glands and sloughing of villus epithelium, and congestion. There was increased urinary space and congestion in the proximal convoluted tubules. Lymphoid follicles increased in size in the spleen, the germinal center enlarged, and congestion in surrounding lymphoid tissue were observed. ConclusionThis study laid a foundation for the research and development of a rapid detection kit for A. pleuropneumoniae and provided conditions for diagnosing and treating porcine contagious pleuropneumonia.

Preparation of a JAZF1 protein polyclonal antibody and its potential role in broiler ascites syndrome

As a novel functional protein, juxtaposed with another zinc finger protein 1 (JAZF1) can regulate the growth and apoptosis through various pathways, and maintain the body's normal physiological metabolism. To explore the important role of JAZF1 in broiler ascites syndrome (BAS), we analysed the expression and distribution of the protein in poultry and mammal tissues based on the prepared polyclonal antibody. In this study, the recombinant plasmid PET32a-JAZF1 was constructed by TA cloning, subcloning and other technical methods, and the fusion protein His-JAZF1 was successfully expressed. After purification, His-JAZF1 was used as the antigen to prepare high-quality chicken-derived antibodies. Subsequently, the results showed that JAZF1 protein in broiler tissues could be specifically recognized by this antibody. Immunofluorescence showed that JAZF1 protein mainly exists in the cytoplasm of pulmonary artery, liver, kidney, heart and lung tissue cells of various animals. The expression of this protein was more obvious in broiler and duck tissues than in mammalian tissues. In addition, western blotting combined with immunofluorescence showed that BAS caused a significant decrease in JAZF1 protein in tissue cells. This effect further indicated that JAZF1 protein was closely related to the occurrence of BAS and provided a new entry point for the functional study of JAZF1 protein.

Prokaryotic expression and polyclonal antibody preparation of human adenovirus type 7 DNA binding protein

Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody. Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA). In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV. Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1 024 000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.