Preparation of a polyclonal antibody against immunogenic fragment of bovine intestinal peptide transporter I (bPepTI) ()
1. INTRODUCTION
Evidence emerged since 1950s that animal intestine can directly absorb peptides of two or three amino acid residues ([1-6]) cloned and analyzed a proton-coupled small peptide transporter called PepTI [6]. This transporter was mainly found in gut. Further studies showed that PepTI was specific to brush border of gastrointestinal absorptive cells [7]. To date, PepTI has been cloned from rats, canines, chickens, humans, rabbits, pigs, ovine and bovine.
It has been found that certain mutations involving malfunction of amino acid transporters result in amino acid deficiency (for example, tryptophan deficiency in Hartnup disease patient), but the symptom can be ameliorated by supplement of dipeptide [8]. Therefore, absorption of small peptide in enterocytes has physiological values, although its nutritional value is unclear. Studies on amino acid flux in gastrointestinal blood stream showed no consistent results. In calves, for example, it has been shown that small peptides constituted 79% of total amino acid absorbed into portal vein in calves [9]. In sheep, however, no small peptide was found to be absorbed into portal vein [10]. Although differences in the results among these studies are attributable to species and diet, more important reason may be due to the inaccurate nature of the flux determination methods. It was clear that the standard errors in measurements of flux differences between portal vein and arteries were huge. Differences among methods in protein preparation led to large differences in observed peptide absorption results [11-13]. The relationship between the level of transporter expression and the amount of substrate supply may, to a certain extent, clarify the importance of small peptide absorption. Studies have shown that small peptides promoted PepTI capacity [14-16] and transporter mRNA levels [17,18]. These results supported the notion that small peptide absorption had nutritional values. However, it was common for transporters to increase expression level when stimulated by their substrates. More convincing evidence may come from PepTI expression elevation seen in glucose transporter under the stimulation of substrate. Unfortunately, no such studies were available to date. Also, changes in mRNA level may not be reflected in protein levels [19]. Studies on whether the substrate stimulates the transporter expression may provide further insight into small peptide uptake.
It is of interest to study how substrate stimulates PepTI mRNA and protein express further. Establishment of an appropriate assay for PepTI protein level is an indispensible step. Protein levels are measured either directly or indirectly. Direct methods require the extraction and purification of proteins directly from tissues. Indirect methods include immunohistochemistry [20], Western blot [7], and ELISA. Indirect approaches require specific antibody. Due to the complexity of protein composition in intestinal mucosa, it is difficult to isolate PepTI. So indirect detection is more applicable. Therefore, it is necessary to prepare antibody. Currently, synthetic peptides have been used for human and mouse PepTI antibody preparation [21,22]. Here, we report the preparation of antibody against bovine PepTI using recombinant bPepTI expressed in prokaryote as antigen.
2. MATERIALS AND METHODS
2.1. Sample Preparation
Healthy Limousin × Luxi hybrid calves were sacrificed in beef processing plant and rumen tissue samples were collected, washed in saline, and then placed on ice-cold glass plate. Rumen epithelial cells were peeled off and transferred to autoclaved 1.5 ml tubes, and frozen in liquid nitrogen. The entire procedure from sacrifice to tissue frozen did not exceed 1 h. The frozen tissues were transferred to lab and store at –70˚C.
2.2. RT-PCR
Total RNA was isolated with Trizol and evaluated with electrophoresis and A260/A280 ratio. Total RNA showed three clear bands in gel. The A260/A280 ratio was between 1.8 and 2.0. These datas indicate the good quality of total RNA. The structure of bPepTI was analyzed with DNA star software. A segment (designated as BP, derived from nucleotide position 1369 - 1695 bp in the ORF) of high antigenicity was selected and amplified following the manual of Takara RNA PCR kit Ver 3.0. The primers were: P1 5’CGCGGATCCCATCGCCATACCCTTCTCGTC-3’, and P2 5’-CCGCTCGAGGGGGCAACCGT TCACTCTTTC-3’. Restriction sites for BamHI and XhoI were separately introduced into the primers. PCR thermal cycles was consisted of pre-denature at 94˚C for 2 min, 30 cycles of amplification (94˚C 30 s, 61.7˚C 30 s, 72˚C 30 s) and a final extension at 72˚C for 2 min.
2.3. Construction and Expression of Recombinant Vector
Construction of prokaryotic expression vector GST-BP. The amplified target sequence was purified, cloned in pMD18-T vector. The vector was propagated in host cell DH5α. Then, the target fragment was transferred to expression vector pGEX-6p-1 after both vectors were double digested with BamHI and XhoI. Recombinant colonies were analyzed with PCR, double digestion and sequencing, which confirmed that the target sequence was correctly inserted in the vector.
Expression and characterization of the recombinant plasmid. The positive plasmid was used to transform host BL21. BL21 is one kind of E.coli bacterial cells. Positive transformant was propagated and induced for 6 h with IPTG at a final concentration 0.1 mM. The resulting culture was disrupted with lysis buffer, lysozyme and ultrasound, examined with SDS-PAGE. The solubility of the expression product was examined. The insoluble portion (inclusion body) was dissolved with urea for further analysis. The soluble portion was analyzed directly.
Western blot analysis of the target peptide. Membrane was blocked with 5% skim milk at 4˚C overnight, incubated with mouse anti-GST monoclonal antibody for 2 h, and then with HRP-conjugated goat-anti-mouse IgG for 1 h. Finally, color was developed by incubation with DAB for 5 - 30 min, and color development was terminated with distilled water.
2.4. Polyclonal Antibody Preparation and Characterization
Immunization. The target protein was isolated with SDSPAGE, negatively stained with pre-chilled KCL (0.25 M) and the target band (white) was collected, freeze-dried, and grinded. The powder was mixed with 1 × PBS of equal volume. The preparation was injected under skin of abdomen and back of mice (100 µg/mouse). Immunization was boosted every two weeks for three times. The negative control mice were injected with saline.
Antiserum collection. One week after the third boost, blood was collected from caudal vein, settled for 1 h at 37˚C and transferred to 4˚C overnight. Serum was separated by centrifugation at 4000 rpm for 10 min, stored at –20˚C until use.
Antibody specificity analysis. The constructed vector pET-32a-BP (His-BP, for short) was expressed in BL21 (DE3), which is one kind of E.coli bacterial cells. The recombinant protein was verified with mouse anti-His tag monoclonal antibody. After verification, the recombinant protein was used for antiserum verification. Specificity of the antibody was analyzed with Western blot, GST-BP fusion protein, GST vector protein, and His-BP fusion protein as antigens. The antiserum was used as the primary antibody.
Antibody titer analysis. The soluble GST-BP fusion protein was used as antigen, and diluted with embedding buffer to final concentrations of 8, 2, 0.5, 0.1 and 0.02 µg/mL. Antiserum and negative serum were diluted to 1:100, 1:200, 1: 400, 1:800, 1:1600. Color was developed with TMB, and stopped by addition of 2 M H2SO4. Absorbance was measured at 450 nm with ELISA reader. And the optimal antigen embedding concentration and optimal antibody dilution were determined [23].
Antigen was embedded at the optimal concentration. Antiserum and control serum were diluted to 1:400, 1: 1600, 1:6400, 1:25600, 1:102400, 1:409600 and 1: 1638400. Optical density was measured at 450 nm with ELISA reader for sample (P), negative control (N), blank control (B). The titer of antiserum was defined as the ratio of (P – B)/(N – B) greater than 2.1.
3. RESULTS
3.1. Amplification of BP Fragment from bPepTI
Sequencing of the amplified product showed that the insert was the target fragment (BP fragment) of 327 bp (Figure 1). Compared with GenBank record (accession BC140526), the amplified BP fragment had three base pair changes: 99 (T-C), 252 (A-G), and 265 (C-T). The first two changes were synonymous and the last change was non-synonymous (Pro89Ser).
3.2. Construction of Prokaryotic Expression Vector
The BP fragment was cloned in pre-defined orientation into pGEX-6p-1 vector. The recombinant plasmids GSTBP and His-BP were double-digested with BamHI and XhoI, resulting in a fragment between 250 and 500 bp in size, which was the expected (Figures 2, 3). These results indicate that the right plasmids were obtained.
3.3. Detection of Recombinant Protein
3.3.1. SDS-PAGE Analysis of Recombinant Protein
Figure 4 showed the expressed recombinant protein in SDS-PAGE gel. Bacterial culture with the null vector pGEX-6p-1 had a 26 kDa band, whereas the culture with the recombinant GST-BP expressed a 38.1 kDa band. The bacterial culture with empty vector pET-32a showed a 18.7 kDa band, whereas the one with His-BP produced a 31 kDa band. These bands were consistent with expected sizes, indicating that the fusion proteins were