The camel immunization was performed according to the protocols with modifications. Healthy male Alxa Bactrian camels raised in the feeding base in Alxa Zuoqi (~3 years old, ~700 kg) were immunized 5 times at weekly intervals with freshly prepared immunogens. 1.0 mg of model antigen, ovalbumin (OVA) was mixed with Freund's complete adjuvant (Sigma-Aldrich, St. Louis, USA) for the first immunization, and with Freund's incomplete adjuvant (Sigma-Aldrich) for the next 4 ones. The prepared immunogens were subcutaneously injected around the bow lymph node in the neck. Blood samples were collected from the jugular vein before the first immunization and 1 day after each week's immunogen administration. Half of the collected blood samples were allowed for clotting at room temperature (RT) for 2 hours. Then the samples were further centrifuged at 5 000 g for 5 minutes at RT to collect the supernatant, and the recovered serum samples were stored at −20 °C for further ELISA to evaluate the serum conversion according to the antibody titers. Another half of the blood samples were collected into the ethylenediaminetetraacetic acid (EDTA)-coated anticoagulant tube and gently inverted to prevent coagulation. Then the samples were further centrifuged at 5 000 g for 15 minutes at RT and the recovered plasma was stored at −20 °C for extraction of total RNA from the peripheral blood lymphocytes (PBLs).
PBLs were isolated via a density-gradient centrifugation method by Ficoll (GE Healthcare, Sweden) according to the manufacture's instruction. The total RNA isolation was immediately performed using the Trizol reagent (Invitrogen, USA) according to the manufactory's instruction. The isolated PBLs was added by 1.0 mL Trizol reagent and then after keeping in RT for 5 minutes, by 200 μL pre-cooled chloroform. Then the mixture was shaken and centrifuged at 12 000 g at 4 °C for 15 minutes and the aqueous phase was carefully collected and transferred to a new tube. Subsequently, 500 μL pro-cooled isopropanol was added into the tube and incubated for 20 minutes at –20 °C. After centrifugation at 12 000 g at 4 °C for 10 minutes, the supernatant was carefully discarded and the pellet was washed with 1.0 mL 75% ethanol. The isolated total RNA was re-dissolved by RNase-free water and the quality was verified by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, USA). The isolated RNA was immediately stored at –80 °C before use.
An aliquot of the obtained total RNA (~500 ng) was reversely transcribed by HiScript II reverse transcriptase (Vazyme Biotechnology, China) in a 10 μL reaction volume according to the manufacturer's instructions. The obtained cDNA was analyzed immediately or stored at –20 °C. Primers used for the quantitation of cDNA specific for camel Th2 cytokines (IL-4, IL-10, and IL-13) were designed according to the sequences from the Genebank database and listed in Table 1. β-actin was used as the internal control. Real-time PCR analysis was performed on the StepOnePlus system (Applied Biosystems, Austin, USA) using ChamQ SYBR qPCR Master Mix (Vazyme Biotechnology) to quantify the cytokine expression level. Briefly, 1 μL of cDNA was added into the 19 μL reaction mixture including 10 μL ChamQ SYBR qPCR Master Mix, 0.4 μL of each primer (to the final concentration of 10 μmol/L), 0.4 μL of ROX reference dye and 7.8 μL of deionized water. The real-time PCR parameters were listed in Table 2. Expression levels were defined through the threshold cycle and the fold changes were calculated according to the equation of 2−△△ct. Each sample was analyzed in triplicate.
Gene Primer (5′→3′) Annealing temperature (°C) Genebank accession number IL4 F: CCCTGGTCTGCTTACTGGTT
55 NM_001303522.1 IL10 F: TCGGAGATGATCCAGTTTTACC
57 NM_001303520.1 IL13 F: GGAGCATCAACCTGACGAC
54 NM_001303521.1 ACTB F: CAGATCATGTTCGAGACCTTC
Table 1. Primers for real-time PCR
Stage Temperature (°C) Time (second) Cycle Hold stage 95 30 1 Cycle stage 95 10 2–41 Annealing temperature 30 Melt stage 95 15 42 60 60 95 15
Table 2. Parameters of real-time PCR and melt curve
To validate the quantification of the expression levels of Th2 cytokines is in accordance with the antibody titer so as to evaluate the precision and versatility of the established method, the antibody titer of immunoglobulin G (IgG) was measured by traditional ELISA. The horseradish peroxidase (HRP) labeled goat-anti-camel IgG was prepared by Prof. Jirimutu's group of Inner Mongolia Agricultural University. A 96-well plate was firstly coated with 100 μL OVA solution (10 μg/mL in PBS buffer) and the plate was then incubated at 4 °C overnight. After washing the unbounded OVA with PBST (PBS buffer containing 0.05% tween 20), the plate was blocked by 2% BSA in PBST buffer at RT for 2 hours. The antisera were serially diluted by 1% BSA in PBS buffer and added into each well at RT, then followed by 10 washes with PBST buffer. Then the plate was incubated with HRP labeled goat-anti-camel IgG at 37 °C for 1 hour. The plate was washed with PBST buffer for 10 times to remove the unbounded conjugate thoroughly and the antibody binding was evaluated by adding 3,3',5,5'-tetramethylbenzidine (Beyotime, China) into each well. The reaction was terminated by adding 2 mol/L H2SO4 and the optical density (OD) value was recorded by a microplate reader at 450 nm (Tecan, Switzerland). The antibody titer was calculated according to the reciprocal of the dilution which has the OD values larger than 0.1 standard deviations above background levels obtained from PBS blank at the same dilutions.
All the data are shown as mean±standard deviation, and the statistical analysis and regression analysis were performed by SPSS 17.0 software and the significance level was set as 0.05.
Pre-evaluation of humoral immune response of Bactrian camels by the quantification of Th2 cytokines using real-time PCR
- Received Date: 2019-03-01
- Accepted Date: 2019-11-13
- Rev Recd Date: 2019-11-05
- Available Online: 2020-01-23
- Publish Date: 2020-11-28
With the increasing immunological studies on camels due to the advantage of their single-chain antibodies for humanizations, it is demanding to develop an easy-to-handle evaluation method of their humoral immune response before proceeding with immunization of foreign antigens that may be toxic to camels. In this study, we quantitatively determined the expression levels of T-helper 2 (Th2) cytokines in peripheral blood lymphocytes obtained from Bactrian camels by real-time PCR. The recorded kinetic profiles resulting from the immunization of ovalbumin (OVA) indicated that after immunization, Th2 cytokines including interleukin (IL) families such as IL-4, IL-10, and IL-13 in the camels were up-regulated by a factor of 1.78, 3.15, and 1.22, respectively, which was validated by traditional enzyme-linked immunosorbent assay (ELISA) methods. Unlike ELISA which requires specific enzyme-labeled antibodies, this established method based on the minimal amount of blood samples holds an advantage in the preliminary evaluation of camel humoral immune response with desirable precision, which is meaningful for biomedical explorations of camel-derived antibodies.
|Citation:|| Xinyu Yu, Yuan Wu, Jiarong Zhang, Jirimutu, Azhati Zulipikaer, Jin Chen.
Pre-evaluation of humoral immune response of Bactrian camels by the quantification of Th2 cytokines using real-time PCR[J]. The Journal of Biomedical Research, 2020, 34(5): 387-394. doi: 10.7555/JBR.34.20190035