cDNA Cloning , Prokaryotic Expression of Two Splicing Products of mLRG , a Mouse Gene of Lipopolysaccharide Response

Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipopolysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns; mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.


Introduction
To date, primary protein lipopolysaccharide responses investigated are lipopolysaccharide (LPS) binding protein (LBP), soluble CD14 and Toll-like receptor 4 (TLR4).The mechanism of these proteins regulate is perplexing.
The binding of LPS to CD14 initiates a wide variety of biological reactions mainly occurring in monocytic cell lineages [1].Since CD14 is not a trans-membrane protein, it couples with the trans-membrane protein, Toll-like receptor 4 (TLR4) and initiates intracellular signaling cascades [2] [3].Soluble CD14 (sCD14) complexed with LPS also activates endothelial cells via TLR4 to induce microcirculatory disturbances [4]- [8].The important roles of CD14 as the LPS recognition molecule in vivo have also been explored using CD14 transgenic and knockout mice [9]- [11].
LPS binding protein (LBP) and CD14 play key roles in promoting innate immunity to Gram-negative bacteria by transferring LPS to the signaling receptor complex, MD-2/Toll-like receptor 4 (TLR4).The dual stimulatory and inhibitory mechanisms of sCD14 and LBP may benefit the infected host by promoting inflammation in local sites, where it is needed, while at the same time preventing potentially detrimental systemic responses to LPS [12].However, the mechanism of LPS-stimulated tissues damage remains obscure.
A number of studies have investigated the impact of LPS and other bacterial products on apoptosis of white blood cells.In vitro, LPS has been shown to promote apoptosis in macrophages [13]- [15] but to inhibit apoptosis of neutrophils [16] [17].In vivo LPS has a pro-apoptotic effect on lymphocytes in Peyer's patches and thymocytes, whereas it has anti-apoptotic effects in peritoneal neutrophils [18]- [20].Mani Alikhani, Zoubin Alikhani et al. demonstrated that lipopolysaccharides indirectly stimulate apoptosis and global induction of apoptotic genes in fibroblasts through experiment, but number of gene participate in this procedure is more than forty [21].So we need to search for upstream gene from cells LPS-stimulated to elucidate the mechanism of LPS damage.
Human lipopolysaccharide response gene (hlrg) was cloned from LPS-stimulated human dental pulp cell by the method of Improved PCR-based subtractive hybridization strategy [22] by Ph.D Chai YB of our teams in 1999 (Accession No. AF143740) [23].We found a more complete novel human lrg cDNA in GenBank database BLAST search (Accession No. AK002071).It may be relative of endotoxin response.Mouse lipopolysaccharide response gene (lrg) is not reported.We compared human lipopolysaccharide response gene (lrg) for mouse ESTs in GenBank database BLAST search [24] and spliced good homology segments to a predicted mouse lipopolysaccharide response gene (lrg) sequence [25].We designed a set of primers at completely homologous fragments in 5'-UTR and 3'-UTR, and amplified two mouse lrg-cDNA sequences by RT-PCR.We got mouse lipopolysaccharide response whole gene (mLRGW) and mouse lipopolysaccharide response splice gene (mLRGS) prokaryotic protein expressed production. Cloning of two splicing products of mouse novel gene mouse lipopolysaccharide response gene (mLRG) and prokaryotic protein expressions are of help in the further study of this gene.

Prediction of Mouse lrg
Comparing human lipopolysaccharide response gene (lrg) to mouse genome with a comparative BLAST genome search and searching completely identical.Splicing some fragments to a whole mouse lrg-cDNA sequence and designing a set of primers at completely homologous fragments in 5'-UTR and 3'-UTR (P1: GCTGCCGTC-ACCTCATGG, P2: TTCACATCAAGGAACCATCG).

Isolation of RNA
NIH3T3 cells were plated on 150-mm glass culture dishes at a density of ~7 × 10 6 cells/plate.Cells were treated with (final concentration 100 μg/ml) lps stimulation for approximately 24 h prior to cell lysis.Cell extracts were prepared at 4˚C; the cells were washed twice with phosphate-buffered saline and lysed in Trizol reagent (purchased from Invitrogen company).Total cellular RNAs were extracted from cells according to the manufacturer' instructions.

Synthesis of cDNA Library and Amply Mouse lrg
The extracted RNA (500 ng) was reverse transcribed into cDNA first-strand using 200 U of Moloney Murine Leukemia Virus Reverse transcriptase (Promega, madison, USA) and 1 mg of oligo (dT) 15 primer (Promega) in a final volume of 25 μl of enzyme buffer (Promega) for 60 min at 42˚C.This cDNA first-strand was taken as template for RT-PCR.PCR was performed with 2 μL of cDNA first-strand and 0.01 pmol of each primer (P1, P2), in a 50 μL reaction volume containing 100 nmol MgCl 2 , 10 nmol dNTPs, 2.5 U of Taq DNA polymerase (Promega), and 5 μL 10 × Taq DNA polymerase buffer.The reaction mixture was heated at 94˚C for 5 min, followed by amplification through 35 cycles.Each cycle included denaturation at 94˚C for 30 s, annealing at 58˚C for 45 s and extension at 72˚C for 90 s.Then 10 μL of the PCR product was electrophoresed on a 10 g/L agarose gel containing ethidium bromide (0.5 g/mL), and visualized under ultraviolet light.

Cloning into Vector and Sequencing
The PCR product was recycled with Plamids Gel Recycle Kit (purchased from BioLabs company), T4 DNA Polymerase (purchased from Takara company) blunt, pUC19 vector was digested using restriction enzyme Hin-cII (purchased from Takara company).The recycled plasmid was inserted in the digested pUC19 vector with T4 DNA Ligase (purchased from Takara company).The ligated plasmids were transformed into engineering bacteria TOP10, the transformed TOP10 was tiled in flat plate contained agar and X-gal.Selecting white colony to sequence.

Cloning to pTAT and Prokaryotic Expression
Designing a set of primers (PF: GCGGGTACCGCGACTCGGCTTGAGGAGGTAACGCGA inlet KpnⅠsite, PR: CCGCTCGAGGCCTTAAGCAGGAGAACC, inlet XhoI site) and amplifying the sequence enconding using LPS-stimulated NIH3T3 cDNA first-strand construct as template and two franking primers(PF, PR), The PCR reaction was performed using standard protocols with 35 cycles of 30 s at 94˚C, 45 s at 53.5˚C and 90 s at 72˚C.Appropriately sized amplification products were verified by agarose gel electrophoresis.The PCR products were inserted in the multiple cloning site of pTAT (Invitrogen) between KpnI and XhoI, transformed into E. coli BL21(DE3) (stored in our labs) respectively.The individual clones were sequenced.The transformant E. coli BL21(DE3) clones were grown at 37.8˚C to an A600 value of 0.6 in LB/ampicillin (100 μg/ml), induced with 1mM isopropyl thio-β-D-galactoside (IPTG) and incubated for 3 h.The induced cells were disrupted by sonication and centrifuged at 12,000 rpm for 10min.Supernatant and sediment were prepared to samples for 12% SDS-PAGE respectively.

Bioinformatics Analysis
The sequences encoding obtained were searched in UCSC database, and analysis chromosome location and exon/intron boundary (http://genome.ucsc.edu/)[26].All exon/intron boundaries were shown to follow the canonical ag/gt rule of splicing.

Cloning Two Splicing Products of mLRG cDNA
The length of applied MLRG cDNA by RT-PCR is 1907 bp (Figure 1), the complete mLRG sequence encoding was known through sequencing, the length of mLRG encoding is 1554 bp (named mlrgW), and we got another splicing form sequence encoding about 1407 bp (named mlrgS), mlrgS lock a fragment about 147 bp between 176 th and 322 th in mlrgW sequence (Figure 2).

Analysis in Murine Genome
We found a g to a change at 229 th nucleotide in the first intron (an amino acid G to D change), a t to g change at 1495 th nucleotide in the tenth intron (an amino acid L to G change), a c to t change at 1566 th nucleotide in the tenth intron, the stop condon antelocate 18 nucleotides through camparing mlrgW and mlrgS to murine genome in UCSC database.mlrgW and mlrgS locate chromosome X qF4 site, mlrgW and mlrgS sequence are 99.8% with homology to murine genome after comparing with sequence in GenBank.Since the genomic organization of mlrgW and mlrgS has not been investigated, we also examined the exon/intron structure of the gene by combining genomic and RT-PCR.The sequence encoding of mlrgW contains 10 exons and 9 introns (Figure 3), and the  sequence of mlrgS contains 11 exons and 10 introns (Figure 4).mlrgS cut out a intron in the second exon of mlrgW, so mlrgS contains 11 exons and 10 introns, it is internal splicing.The open reading frame (ORF) of these two are correct.It demonstrates that these two splicing products of mlrg are correct different expressions of murine lrg (Figure 5).They maybe acted as two different roles in responsing to LPS-stimulate.

Prokaryotic Protein Expression
mlrgW and mlrgS were cloned to pTAT vector successfully, and results of sequencing were identified with sequence results of mlrg-cDNA, pTAT-MLRGW and pTAT-MLRGS were identified using cut with double enzymes (KpnI and XhoI), monoenzyme (HindIII) respectively (Figure 6). Figure 7 shows that mlrgS was divided into two fragments (one is about 680 bp, another is about 730 bp) by HindⅢ.It is not obvious in Agarose gel electrophoresis because two fragments is too close.All identified restriction sites are correct.It demonstrated that mLRGW-pTAT/ BL21(DE3) and mLRGS-pTAT/BL21(DE3) we cloned were right, and can be used to express mLRGW and mLRGS proteins correctly.His-TAT-mLRGW protein contained 468 amino acids band at Mr 66500, and His-TAT-mLRGS protein contained 518 amino acids band at Mr 61,000.The result is concordant with predicted molecular weight.His-TAT-mLRGW and His-TAT-mLRGS were existed in inclusion body.His-TAT-mLRGW and His-TAT-mLRGS were not expressed in mLRGW/BL21(DE3) and mLRGS/BL21-(DE3) without induction.The result demonstrates that mLRGW/BL21(DE3) and mLRGS/BL21(DE3) were inducible (Figure 8).Further bioinformatics analysis shows mlrgW and mlrgS contain a Leucine Zipper domain between 431 th and 468 th amino acids motif at mlrgW and a domain between 606 th and 807 th nucleotide motif at mlrgW matches Pfam-B 27780 associated with Zinc Finger Protein (ZIP) C 2 H 2 type in the Protein families database (Pfam database).The GAG (=CTC) repeat among Zinc Finger Protein (ZIP) C 2 H 2 type constitutes a high-affinity site for Sp1 binding to the wt1 remoter.These manifest that mlrg maybe involved in transcription regulation.Leucine zipper domain is the property domain of DNA binding protein [27], but it is riddle which proteins mlrg bind with.
Former studies show over-expression human lrg can inhibit at G 0 stage of cell cycle in lrg-transfected human embryo kidney cells and HepG2 cells.mlrg maybe involved in the procedure of oxidative stress, and then impact cell cycle.We are going to demonstrate the mechanism of lrg impact cell cycle through further experiment.

Figure 1 .
Figure 1.Agrose gel electrophoresis of the PCR product of mLRG cDNA.M: 2000 bp ladder marker; 1: the PCR product of mLRG cDNA.

Figure 2 .
Figure 2. ORF sequence and the deduced amino acid sequence of mLRGW and mLRGS cDNA.

Figure 3 .
Figure 3. Exon/intron structure of mlrgW.Exon bases are given in capital letters, intron bases in small letters.Canonical ag/gt bases at the exon/intron boundaries are underlined.

Figure 4 .
Figure 4. Exon/intron structure of mlrgS.Exon bases are given in capital letters, intron bases in small letters.Canonical ag/gt bases at the exon/intron boundaries are underlined.

Figure 5 .
Figure 5. Genomic structure of mLRGW and mLRGS.Introns are represented by a thin line.Exons are depicted by a filled box.(a) Represents mLRGW; (b) Represents mRLGS.

Figure 6 .
Figure 6.Agarose gel electrophoresis of the PCR product of mLRGW and mLRGS cDNA ORF.M: DNA ladder marker; 1: the PCR product of encoding the mLRGS protein; 2: the PCR product of encoding the mLRGW protein.