Construction of CTLA-4-Ig Fusion Gene in pBudCE4.1 Expression Vector.

BACKGROUND: CTLA-4 inhibitory signals prevent cell cycle progression and IL-2 production, leading to a halt on an ongoing immune response. CTLA4-Ig fusion proteins contain the extracellular domain of CTLA-4 and Fc fragment of human IgG antibody. In this study we aimed to fuse the ctla-4 gene encoding the extracellular domain of CTLA-4 molecule with igg1 gene encoding Fc region of human IgG.
METHODS: After primer design, PCR reaction was performed using pfu polymerase enzyme and specific primers. PCR amplified fragment was ligated into the vector containing the human igg1 gene. The resulting fusion fragment of ctla-4 and human igg1 genes was ligated to pBudCE4.1 expression vector.
RESULTS: Extracellular domain of ctla-4 gene was ligated to igg1 gene and then ctla4-ig fragment was cloned into pBudCE4.1 vector. Construction of the expression vector was confirmed by restriction pattern analysis and sequencing.
CONCLUSION: By confirming the construct, in the next step, the recombinant DNA will be used to produce CTLA4-Ig recombinant protein for the clinical uses.

Introduction

Following the T cell activation, cytotoxic T lymphocyte antigen-4 (CTLA-4), a negative regulatory molecule, will be expressed on T cells [1,2]. CTLA-4 is a homolog of CD28 but binds B-7 molecules with greater affinity. Inhibitory signals of this molecule inhibit cell cycle progression and IL-2 production, leading to a halt on an ongoing immune response [3-6].

Regarding the central role of CTLA-4 in down-regulation of the immune responses, co-stimulatory receptors became an important target for drug development. The best example is the CTLA4-Ig fusion protein, containing the extracellular domain of CTLA-4 and the constant region of human IgG antibody. This fusion protein can inhibits T cells dependent immune responses [5,7,8].

In this study we aimed to fuse extracellular domain of ctla-4 gene to Fc region of human igg1 gene. This recombinant DNA could be used to produce CTLA4-Ig protein and studding its function in future studies.

Materials and Methods

Enzymes and chemicals

All chemicals and antibiotics were purchased from Sigma, Merck (Germany) and Invitrogen (France), unless stated otherwise. DNA-modifying enzymes and restriction enzymes were obtained from Fermentas.

Vectors, microorganisms and growth conditions

Escherichia coli DH5α (CinnaGen, Iran) as a host and pBudCE4.1 as an expression vector were used. IgG1 containing vector was provided kindly by Dr. Rabbani (Avicenna Research Institute, Iran). Escherichia coli (E. coli) were cultured in LB medium at appropriate temperature (37°C) with shaking (150 rpm).

PCR amplification and CTLA4-Ig fragment construction

Extracellular domain of ctla-4 gene was amplified using specific primers (CTLA4-FOR/CTLA4-fuse) and pUCCTLA4 vector as template. For subsequent cloning of the PCR-derived fragments, SalI and BamHI restriction sites were added to the 5′-end of these primers, respectively (Table 1). pUCCTLA-4 (synthetic construct) was used to amplify the ctla-4 gene with pfu polymerase enzyme. PCR products were purified by High Pure PCR Product Purification Kit (Roche, Germany). The purified fragment was digested simultaneously with vector containing human igg1 gene using SalI/BamHI enzymes and then were ligated. E. coli DH5α cells were transformed using CaCl2 method [9]. Recombinant colonies were confirmed by PCR using specific primers. Plasmid DNA preparation was done using QIAGEN Mini Prep Kit (Germany).

Table 1.

Oligonucleotides (primers) used in present study (restriction sites were showed in bold)

Primers	Sequence 5′ to 3′	Orientation	5′ cloning site
A) Primers used for amplification of ctla4-Ig
CTLA-4 FOR	5′-TTGTCGACAGCCACCATGGCTTGCCTT-3	Sense	SalI
CTLA-4 fuse	5′-TTGGATCCGTCAGAATCTGGGCA-3′	Anti-sense	BamHI
B) Universal primers
T7f	5′ GTAAAACGACGGCCAGT	Sense	--
pBudCE4.1r	5′ CAGGAAACAGCTATGAC	Anti-sense	--
M13f	5′-GTAAAACGACGGCCAGT-3′	Sense	--
M13r	5′-AACAGCTATGACCATG-3′	Anti-sense	--

Construction of CTLA4-Ig expression vector

Vector containing the ctla4-ig fragment digested by SalI/XbaI enzymes and subcloned into pBudCE4.1 SalI/XbaI cloning sites with the methods mentioned before. Recombinant colonies were confirmed by digestion with cloning enzymes and PCR pattern.

Sequence and computer analysis

Cloned DNA fragment in pBudCE4.1 (50–200 ng/μl) was sequenced by a Commercial Service (Bioneer, South Korea).

Results

ctla-4 fragment was amplified using specific primers and pfu polymerase. A specific band about 483 bp showed the expected size (Figure 1). Extracellular domain of ctla-4 gene was inserted into the SalI and BamHI pGEMIgG vector and designated as pGEMC-TLA4-Ig. The new construct was confirmed by restriction pattern using SalI/BamHI and SalI/XbaI enzymes and PCR product pattern. According to the size of ctla-4 gene external domain (483 bp) and igg1 gene (993 bp), the resulting fragment (1476 bp) confirmed the fusion of extracellular domain of ctla-4 to human igg1 gene (Figure 2).

Figure 1.

ctla-4 gene PCR product with Pfu polymerase enzyme M: 1 kb ladder

1: PCR product using specific primers (CTLA4- FOR / CTLA4-fuse).

Figure 2.

Confirmation of new construct with digestion pattern

A) 1: Digestion of vector containing igg1 gene with SalI/XbaI enzymes

2. Digestion of new construct with SalI/XbaI enzymes

B) 1: Digestion of new construct with SalI/BamHI enzymes.

To clone ctla4-ig fragment in pBudCE4.1 expression vector, pGEMCTLA4-Ig construct was digested and gel purified fragment was cloned into pBudCE4.1 vector. Construction of the expression vector was confirmed by restriction pattern analysis using SalI and XbaI. The cloned fragment was sequenced by T7f/p-BudCE4.1r universal primers. DNA sequencing showed an open reading frame, 1476 bp in length, encoding a 492 amino acid polypeptide. The new construct designated pBudTJ1 is shown in figure 3.

Figure 3.

pBudTJ1 schematic view.

Discussion

Co-stimulatory molecules play a critical role in controlling the immune response. The central role of CD28 family, especially the CTLA-4, makes it a useful tool for immunotherapy in autoimmune disease and transplant rejection [6,10].

Two approaches have been selected in respect to the potential clinical applications of CTLA-4 in immunotherapy, anti CTLA-4 antibody and CTLA4-Ig, respectively [7,11]. CTLA4-Ig is a fusion protein containing the extracellular domain of CTLA-4 and the Fc portion of human IgG1. This protein is capable of preventing the stimulatory effect of CD28 through competing and binding to B-7s on APCs [12-14].

In this study, we have fused the extracellular domain of CTLA-4 to the Fc fragment of the human IgG1 antibody. The resulting construct was ligated to pBud-CE4.1 expression vector and confirmed by sequencing. Analysis of the fusion sequence revealed an open reading frame encoding a protein of 445 amino acids with predicted molecular mass of about 50 kDa without glycosylation (ExPASy). In subsequent study, this recombinant DNA can be used to produce CTLA4-Ig recombinant protein.