Abstract

CCRL1 encoded by gene belongs to the family of G-protein coupled receptors. This protein is a kind of C-C chemokine receptors which has a basic role in evolutionary function of heart identified chemokines. The aim of this study is to improve the creation of sticky ends at CCRL1 gene by T-vector. In this study, CCRL1 gene was amplified by PCR. Restriction sites of BamHI and NotI enzymes were introdused into the 5′end of the forward and reverse primers, respectively. Mentioned enzymes are in the endonuclease family group therefore can not create sticky ends at both sides of CCRL1 gene to overcome this problem amplified fragment by Taq polymerase enzyme was inserted into T-vector. Then, recombinant vector was transformed in E. coli.

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INTRODUCTION

Seven trans-membrane receptors are a group of proteins with seven helical regions located in cell membrane (Khoja et al., 2000). Helical regions include 20-30 aminoacids that linked with three extracellular and intracellular loops.

These receptors have an extracellular amino-terminal and an intracellular carboxy-terminal. Since, these receptors adjoin to G-proteins, those are also named G-Protein Coupled Receptors (GPCR). These receptors only exist in eukaryotic cells (King et al., 2003). Several factors as neurotransmitters, peptides, ions, hormones and ligands are bound to these receptors and lead to signal transduction within these channels. Amino acids in helical regions of these receptors are highly conserved (Joost and Methner, 2002). The GPCRs are categorized to 100 sub-families according to sequence similarity and ligand structure and function.

Despite of sequence differences in GPCR’s subgroups, all of them have same structure and signal transduction mechanisms.

Some of the used bioinformatics methods for classification of GPCRs are just based on the similarity of their amino acid sequence (Xiao et al., 2009; Qiu et al., 2009; Gu et al., 2010). In this study, we describe the optimization the creation of sticky ends at both sides of CCRL1 gene by T-vector.

MATERIALS AND METHODS

RNA extraction and cDNA synthesis: Human spleen tissue was provided from accidental patients. The 30 mg of the spleen tissue was homogenized and RNA was extracted by RNeasy mini kit (QIAGEN). Extracted RNA was dissolved in distilled water. To ensure the integrity of extracted RNA, 2 μL of each sample was electrophoresed and then the Optical Density (OD) of each sample was read out at 260 nm. At next stage total cDNA was synthesized from extracted RNA by cDNA synthesis kit (Fermentas). To confidence the accuracy of cDNA synthesis, PCR reaction was performed on each sample for amplification of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) House keeping gene.

Amplification of human CCRL1 gene: In order to design specific primers, human CCRL1 gene was deduced from NCBI (Accession number: AY221094.1) and analyzed with oligo6 software. The restriction sites of BamHI and NotI enzymes were designed at 5'end of forward and reverse primers, respectively.

The sequence of forward and reverse primers is 5′-AA AGGATCCGCTTTGGAACAGAACCAGTCA-3′ and 5′-GG GGCGGCCGCTTAAATGCTAAAAGTACTGGT-3′, respectively (restriction sites are shown by italics). Primers were ordered from Metabion Company (Germany). To amplify the CCRL1 gene, PCR reaction was performed as follow: 0.3 μL of Smar Taq Polymerase (Stock: 5 U μL^-1), 0.5 μL dNTP (stock: 10 mM), 0.5 μL from each forward and reverse primers (stock: 100 pM), 0.75 μL MgCl₂ (stock: 50 mM), 2.5 μL 10X PCR buffer and 2 μL cDNA (1000 ng mL^-1). Final volume of 25 μL reached by addition of ddH₂O in the PCR mixture. (All PCR reagents were purchased from Fermentas). The reaction was done by thermocycler (Astec) as follow: Initial denaturation at 94°C for 5 min was followed by 30 repetitive cycles including denaturation temperature: 94°C for 1 min; annealing temperature: 56°C for 1 min, extension temperature: 72°C for 1 min. The amplified product was precipitated and dissolved in ddH₂O for further applications.

Insertion of amplified fragment into T-vector: The 1 μL of amplified human CCRL1 gene was added to 10 μL from T-vector solution kit (Fermentase). For ligation purposes, this mixture was placed at 16°C for 12 h. Then, the mixture was transformed to E. coli cells. Transformed E. coli was cultured on media containing X-gal and IPTG. Recombinant vector was extracted from white colonies. Extracted vector was digested by BamHI and NotI enzymes according to the protocol.

RESULTS AND DISCUSSION

Amplification of CCRL1: As shown in Fig. 1a, the total spleen RNA, loaded on agarose gel, composed of 28, 18 sec and degraded RNA bands. The cDNA synthesis was approved by GAPDH amplification (Fig. 1b). Using the synthesized cDNA as template, CCRL1 gene containing fragment with the approximate size of 1053 bp was amplified by utilizing the conditions (Fig. 1c).

Digestion of recombinant T-vector: Smar Taq polymerase adds adenine nucleotide at the 3′ end of amplified fragment. Therefore, amplified fragment can insert into T-vector. Whereas, BamHI and NotI are endonuclease enzymes so will be more effective on inserted fragment into T-vector. As shown in Fig. 2 CCRL1 fragment was cut from T-vector with a suitable concentration.

In many studies was used T-vector. For example Shizuya and Goda were used T-vector in their researches (Goda et al., 2004; Shizuya et al., 1992). Hamiduddin determined the various expression level of this gene in different tissues using Northern blot. The maximum expression was demonstrated in heart tissue.

It is also express in other tissues as lungs, pancreas, spleen, ovaries, small intestine, fetal lung, liver and kidneys (Khoja et al., 2000). In this experiment, due to lack of access to human heart tissue, we have isolated CCRL1 gene from human spleen. We used E. coli (DH5α) cells for amplification of T-vector. Yung-Jiang have cloned PACAP gene, belonged to the GPCR into the pAlter1 vector and created site-directed mutagenesis in PACAP gene. As this study, they have amplified their recombinant vectors in E. coli bacteria (DH5α) (Yong-Jiang et al., 2000). Jo and Villalta were used NcoI, HindIII, EcoRI, BglII, PvuII, BstEII and PstI in their researches whereas we use BamHI and NotI in the research (Jo and Jo, 2001; Villalta et al., 1992).

Fig. 1:	Experimental steps for constructing CCRL1. a) RNA isolation from spleen tissue. b) RT-PCR product comprising the GAPDH cDNA which is shown by arrowhead. c) Agarose gel mobility of whole length of CCRL1 gene, the amplified band is indicated by arrowhead. M: 1k bp DNA ladder

 

Fig. 2:	Digestion of T-vector by BamHI and NotI. The CCRL1 fragment and T-vector is shown by arrowhead

 

CONCLUSION

In this study, bacteria that had received the recombinant vector formed white colonies on media containing X-gal and IPTG whereas other bacteria formed blue colonies. Recombinant vector was extracted and easily was digested by mentioned enzymes.

How to cite this article:

Mohammad Karimian, Mohammad Hashemi, Mohadese Behjati and Abolghasem Esmaeili. Optimization the Production of the Sticky Ends of Human CCRL1 Gene.
DOI: https://doi.org/10.36478/jmolgene.2011.5.7
URL: https://www.makhillpublications.co/view-article/2070-4267/jmolgene.2011.5.7