| BackgroundThe bowel injury is closely associated with dysfunction of intestinal mucosal barrier. Especially, the importance of immune barrier and biological barrier in intestinal mucosa have been gradually realized in recent years. Inflammatory bowel disease (IBD) are a group of chronic intestinal Inflammatory deseases including Crohn's disease (CD) and ulcerative colitis(UC). Generally speaking, IBD is associated with genetic background, disorder of immunoregulation, individual difference and environment in etiology, and finally demonstrate the mucosal injury resulted from the abnormity of the immune-reaction between intestinal mucosa and foreign microorganism antigen. At the present time, incidence of IBD is increasing, and the life quality of the patients with IBD is badly affected. The traditional therapeutic strategy is to aim at intestinal microorganism infection and activation of intestinal immune system, patients are treated with the nonspecific anti-inflammatoy drugs and immunosuppressive agents mainly, antibiotics additionally. However, the side effects and toxic reactions are evident. Especially there is no effective method to cure refractory CD cases, and the natural course of CD is unable to change.By researching on biological therapy for CD, the effective modulatory therapy has been discovered ,which is in allusion to the balance among various inflammatory cytokines. Nevertheless, we should pay attention to their exact therapeutic effects and biosafeties.As recent studies indicated, the pathological mechanism of CD is possibly related to functional defect of intestinal innate immune defense, neutrophils and macrophages. Furthermore, recombinant human granulocyte colony stimulating factor (rhG-CSF) and recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) have been used to treat CD via enhancing neutropil chemotaxis and the functions of monocytes-macrophage to modulate intestinal innate immunity. As far as we know, rhGM-CSF used in clinics or studies is obtained from constructed recombinant plasmids composed of cDNA encoding hGM-CSF and its corresponding expressive vector and transforming them to Escherichia coli, yeast, attenuated salmonella, et al. However, the security should be under watch because they are administrated by hypodermic and several side effects are discovered.The mucosal immunity and systemic immunity of the intestine are modulated by defferent groups of commensal bacteria in the intestine. Lactococcus lactis (Streptococcus lactis) are commensal bacteria in gut and are considered GRAS (generally regard as safe). They can enhance the function of intestinal mucosa barrier by ameliorating gastrointestinal microeubiosis, inhibiting the adhesion of intestinal pathogenic bacteria and the intestinal mucosal susceptibility to bacteria translocation. And that, they are heavily used for the delivery of recombinant polypeptides or proteins because they do not produce LPS (lipopolysaccharide) or any proteases as Escherichia coli do and Lactococcus lactis rebuilt is able to endure bile and acid. Recently, Steidler L et al and Vandenbroucke K etal have successfully constructed recombinant Lactococcus lactis which can secretorily exppress bioactive rhlL-10, rhTFF respectively in colon and cure experimental colitis. However, there is no report about recombinant hGM-CSF in Lactococcus lactis so far.ObjectiveTo construct .recombinant Lactococcus lactis which would secretorily express bioactive hGM-CSF protein in colon and be applied to the therapy for CD via oral administration in future. It might utilize the protective effect on intestinal mucosa of hGM-CSF and Lactococcus lactis. Additionally, it would have the following advantages such as locally expressing bioactivity, having not any antigenicity, using safely, being convenient to orally administration , being convenient to largely produce. Thus, it would be likely to exhibit better treatment effect. Main contents (Methods and Results)According to sequences published in GenBank, Lactococcus lactis secretion expressive system was designed, hGM-CSF gene coding was optimized and hGM-CSF cDNA was synthesized via PCR based gene assembly(PBGA) and gene cloning technology. Lactococcus lactis secretion vector pTRCSF was construvted and recombinant Lactococcus lactis LL-CSF was obtained. Moreover, Lactococcus lactis secretion expressive vector pTRCSFGFP and recombinant Lactococcus lactis LL-CSFGFP were constructed in order to identify whether recombinant Lactococcus lactis LL-CSF is able to located in intestine and secretorily express hGM-CSF pretein. Then, expression of hGM-CSF pretein and its bioactivity in recombinant Lactococcus lactis LL-CSF were determined with cellular experiment in vitro. Finally, colitis-induced by trinitrobenzenesulfonic acid (TNBS) in rats was preparaed to evaluate the protective effects of LL-CSF on experimental colitis for that the pathology mechanism and histopathology alterations of colitis-induced by TNBS in rats are similar to human CD.â… . Construction of recombinant hGM-CSF Lactococeus iaetis1. Construction of Lactococcus laetis secretion expressive systemAccording to sequences publicated in GenBank, we chose the Lactococcus lactis subsp. Cremoris promoter59(p59) as the promoter, chose the ribosome binding site(RBS) and the gene coding signal peptide of Lactococcus lactis secreted protein Usp45 as RBS and signal peptide(SPusp45) gene, multiple cloning site(MCS) was inserted after the SPusp45, the terminator of Usp45 gene(T-usp45) was used as the terminator. PCR(Polymerase chain reaction) based on gene assembly was used to synthesize the gene PUS containing P59 promoter, USP45 RBS, SPusp45 gene, T-usp45 and MCS. Then, PUS gene was ligated to vector pGEM-T easy by TA cloning and the complete correct PUS sequence was obtained after sequencing. Furthermore, PUS sequence was subcloned to pBluescriptâ…¡SK (＋) and plasmid pPUS containing Lactococcus lactis secretory expressive system was acquired. And the result of sequencing was completely in line with our design.2. To assemble hGM-CSF gene to express in Lactococcus lactis in vitroBased on the amino acid sequence ofhGM-CSF (GenBank No:AAA52121) and the protein expression preferable codon of Lactococcus lactis, the entire human hGM-CSF gene condon sequence was designed to 16 complementary oligonucleotides instructed by DNAWORKS program on line, and restriction enzyme sites of Pstâ… and BamHâ… were added in number 1 and 16 oligonucleotide respectively, hGM-CSF cDNA was obtained by PCR based on gene assembly. Then hGM-CSF cDNA was cloned to pGEM T easy vector. The positive colonies were confirmed by restriction enzyme excision and sequencing.3. Construction of Lactococcus lactis secretion expression vector pTRCSF and pTRCSFGFPFirstly, plasmid hGM-CSF obtained from TA cloning and plasmid pPUS were respectively excised by restrictive enzyme-reaction with PstI and BamHI, and the optimized hGM-CSF seqence was to cloned to the plasmid pPUS which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 terminator to obtain plasmid pPUSCSF, and identified by seqencing. Meanwhile, the up-primer and down-primer were designed to amplify EGFP by PCR and the positive sDGFP plasmid was obtained by TA-clone and verified by DNA sequencing. Then, the positive sDGFP plasmid was cloned to pPUSCSF to get plasmid vector pPUSCSFGFP. Finally, after double restrictive reaction with XbaI and SacI, pPUSCSF and pPUSCSFGFP were cloned to shuttle vector pTRKH2 to acquire Lactococcus lactis secretory expressive vector pTRCSF and pTRCSFGFP.4. Construction of LL-CSF and LL-CSFGFPLactococcus lactis 1403 competent cells were prepared at first. Then plasmid pTRCSF and pTRCSFGFP were transformed in LL-1403 by electroporation to obtained LL-CSF and LL-CSFGFE Recombinant Lactococcus lactis LL-CSF and LL-CSFGFP were successfully constructed which was verified by restrictive enzyme-reaction with XbaI and SacI to plasmids extracred from LL-CSF and LL-CSFGFP. As a control, the same method was used to turn pTRKH2 into LL-1403 to construct LL-pTRKH2.â…¡. hGM-CSF protein expressed in LL-CSF and its bioactivity determination The total proteins and supernatant proteins of LL-CSF, LL-pTRKH2 and LL-1403 were extracted to observe the expression of hGM-CSF protein through SDS-PAGE and Western Blot. Then, we investigated the multiplication effects of supernatant fluids of LL-CSF, LL-pTRKH2 on TF-1 cell strain which is growing depends on hGM-CSF. Then, hGM-CSF protein bioactivity in LL-CSF was determined by using CCK-8(cell counting kit-8). The Leubene, a kind of homemade rhGM-CSF , being used in clinical therapy was taken as positive control, Cellular cultivation fluid RPMI 1640 was taken as negative control.1. Expressions of hGM-CSF protein in recombinant hGM-CSF Lactococcus lactis10μ1LL-CSF, LL-pTRKH2 and LL-1403 GM17 stock fluids were incubated into culture solution of 2ml GM17. The bacteria were cultured in 30℃for 24 hours, ended at 0.8 OD 600nm. The total proteins and supernatant proteins of LL-CSF, LL-pTRKH2 and LL-1403 were extracted to observe the expression of hGM-CSF protein through SDS-PAGE and Western Blot. The obvious extrinsic protein belt about 16.4kDa(hGM-CSF protein 14.4kDa added USP protein) was found both in supernatant proteins and total proteins of LL-CSF by PAGE-gel, and the specific protein blot was found in the corresponding site via Western Blot with anti-hGM-CSF monoclonal antibody. There was not any extrinsic protein belt found in supematant proteins and total proteins of LL-pTRKH2 and LL-1403. These results indicated that the recombinant hGM-CSF Lactococcus lactis LL-CSF constructed in this study could secretorily express hGM-CSF protein in vitro.2. Determination of protein bioactivity of recombinant hGM-CSF Lactococcus lactis LL-CSFLL-CSF and LL-pTRKH2 was cultivated in RPMI1640, the supernatants were filtered to utilize in observing their effects on proliferation of TF-1 cell strain which depends on hGM-CSF. Moreover, hGM-CSF protein bioactivity in LL-CSF was determined by using CCK-8(cell counting kit-8). The Leubene, a kind of homemade rhGM-CSF ,being used in clinical therapy, was taken as positive control, cellular cultivation fluid RPMI 1640 was taken as negative control.(1) The cell cultivation and observation of the multiplication TF-1 cell strain was descent-cultivated in cellular cultural fluid which containing 20ng/ml rhGM-CSF(Leubene), RPMI1640, 10%FBS(fetal calf serum), 10% Pen/Strep. The well-developing TF-1 cells were syringed by RPMI1640 without serum for three times. Then, they were added to six vessels filled with 30ml the above cell-cultural without Leubene fluid. Moreover, several kinds of different volume of supernatant of LL-CSF and LL-pTRKH2 growing in 0.8 OD600nm, 4μ1Leubene were respectively added in them, another vessel only uses RPMI1640 cell culture fluid. Then, they were put into incubator to culture. TF-1 cells in culture flasks with LL-CSF or Leubene appeared evident proliferation by observing under inverted microscope after 48 hours. It indicated that recombinant Lactococcus lactis LL-CSF had the bioactivity of hGM-CSF protein.(2) Determination of protein bioactivity of recombinant Lactococcus lactis LL-CSF Leubene (rhGM-CSF) was diluted to 25ng/ml with cellular cultural fluid containing RPMI1640, 10%FBS,10%Pen/Strep, LL-CSF supematant fluid was diluted to 5μ1/ml by the same way. Each 100μ1 of them was respectively added into 96 wells plate respectively after gradingly diluted by time-ratio. TF-1 cells growing in log phase were washed by PBS for three times and suspended in cellular solution containing RPMI1640, 10%FBS, 10%Pen/Strep, then these TF-1 cells were modulated to 2×104CFU/ml and each 100μl of them was incubated in 96 wells plate respectively. The 96 wells plate was put in incubator to culture at 37℃, 5%CO2 for 48 hours. After each 20μl of CCK-8 reagent was added into every well, the 96 wells plate was continue to incubated for 2 hours. Furtherly, OD450nm valueswere determined by enzyme-labeled apparatus. Finally, the bioactivity of 5μl LL-CSF supernatant fluid was corresponded to the specific activity of 18ng rhGM-CSF Leubene via reckoning.â…¢. The protective effect of LL-CSF on TNBS-induced colon damage in ratsFirstly, eight SD rats fed with LL-CSFGFP were to observe expression of EGFP pretein of membrane bacteria in colon so as to verify whether recombinant Lactococcus lactis LL-CSF is able to located in intestine and express heterologous pretein secretorily. Then, trinitrobenzenesulfonic acid(TNBS) was used to prepare the animal model of colitis in SD rat. And that the pathological mechanism and histopathological alterations of colitis-induced by TNBS in rats are similar to human CD. Basing on the well-known pathological course of CD in combination with our results studied above, the time periods of intervention and observation were determined. Then, the general states, the body weight change, the pathological score and the expression of some related cytokines were compared with each group of rats for discussing the protective effect of LL-CSF on the experimental colon damages induced by TNBS in rats.1.Validation on the location and expression of recombinant hGM-CSF Lactococcus lactis in colon of rats Each two of the eight SD rats(male and female, each rat weighs about 180g were fed with lml LL-CSFGFP growing in log phase about 0.8OD600nm(about 5×109CFU) on the first, third, fifth, seventh day respectively. All rats were killed on the eighth day and 2g colon tissue of each rat was to culture membrance bacteria. Lactococcus lactis screened out from the plates with erythromycin-resistence were to be observed the expression state of EGFP by Laser scaning confocal microscope. There were some flora and expression of EGFP in rats fed with LL-CSFGFP on the seventh day, there were fewer flora and no expression of EGFP in others. It identified that recombinant hGM-CSF Lactococcus lactis we constructed are able to located in colon of rats and possibly express hGM-CSF pretein secretorily, but the stability should be studied further.2. Preparation of the animal model of colitis in SD rat and grouping32 cleaning-grade SD rats(each weighs about 180g, half male and female) were routinely fed for a week. After fasting of a day, they were randomly divided into normal control, normal saline(NS) group, LL-pTRKH2 group, LL-CSF group. Then 0.9ml 2.5%TNBS (diluted in 50% ethanol) were respectively administered by anus for each of the rats except normal control after the rats were respectively anaesthetized with 0.1 ml 3% pentobabital sodium by peritoneal injection. They were given routine feeding after 10 hours. Finally, 1mlNS, 1ml(5×109 CFU) LL-pTRKH2, lml LL-CSF were respectively orally administrated to rats of the later three groups per day since the second day after TNBS treated.3. Observing on the general states of rats and collection of colon speeimensThe general states of rats such as spirits, eating, activity, faeces were recorded in everyday. It is found that the general state and the body weight loss of rats in LL-CSF group were obviously improved compared with others treated by TNBS. The rats were killed on the eighth day. After the states of pathological changes in peritoneal space, colon dimensions and serosa lesions had been observed, 10cm colon tissues lcm far from anus were extracted and the intestinal contents were washed by NS. Gross pathological changes were detected after washed by NS. Colon tissues in lesions areas or surrounding by lesion areas or the same parts were taken to observe the gross score, the histopathological score as well as the protein expression of COX-2 and PCNA, the mRNA expression of IL-6, IL-18, IL-12p35, IL-12p40, IFN-γ.4. Observations on main indexes(1) Pathological changes score By gross observation, it was found that there were different extents of exuding or adhesion in abdominal cavity, colon expanding and congesting, hemorrhage, erosion/necrosis or ulcer in colon mucosa in all rats TNBS treated. By histopathology observation on colon tissues of rats TNBS treated, the mucosal epithelial cells suffered injury, there were mocosa edema, hemorrhage, the crypt becominging shallow or crypt abscess, and inflammatory cells infiltrating in submucosa et al. The gross pathological score and histopathology score of LL-CSF group were both obviously lower than other groups TNBS treated(P＜0.001). It indicated that LL-CSF had apparent protectiv effect on TNBS-induced colitis in rats.(2) Protein expression of COX-2 and PCNA in colon tissues of rats As observed by immunohistochemical method, the protein expressions of COX-2 and PCNA in colon tissues of rats in normal control were respectively negative and weak-positive, the protein expressions of COX-2 and PCNA was found in others. The protein expression of COX-2 in LL-CSF group was positive and protein expressive level of PCNA was strong-positive. The protein expressions of COX-2 in. NS group and LL-pTRKH2 group were both strong-positive, the protein expressions of PCNA in NS group and LL-pTRKH2 group were positive, That further indicated that LL-CSF had evident protectiv effect on TNBS-induced colitis in rats.(3) The expressions of IL-6, IL-18, IL-12p35, IL-12p40, IFN-γmRNA in colon tissues of rats The expressions of IL-6, IL-18, IL-12p35, IL-12p40, IFN-γmRNA in colon tissues of rats were determined by RT-PCR(reverse transcription polymerase chain reaction). The expressive levels of IL-6, IL-18, IL-12p35, IL-12p40, IFN-γmRNA of colon tissues in rats TNBS-treated were obviously higher than those of nomal control(P＜0.001). Moreover, the expressive levels of IL-6, IL-12p35, IFN-γmRNA of colon tissues in LL-CSF group were evidently lower(P＜0.001, P＜0.001) than those of other two groups TNBS-treated. However, there was no obvious differencs of the expressive levels of IL-18, IL-12 p40mRNA of colon tissues among three TNBS-treated groups(P＞0.050).The results of animal experiment indicated that cytokines IL-6, IL-18, IL-12p35, IL-12p40, IFN-γtook part in the pathological mechanism of TNBS-induced colitis in rats, TNBS-induced colitis in rats was Thl-associated colitis which was similar to human CD, LL-CSF was of obvious protective effect on TNBS-induced colitis in rats which might be related to down regulation of cytokines IL-6, IL-12p35, IFN-γmRNA.Conclusions1. hGM-CSF cDNA and Lactococcus lactis secretion expressive system gene PUS were successfully assembled.2 Lactococcus lactis secretion expressive vector pPUS were successfully constructed.3. Recombinant hGM-CSF Lactococcus lactis cloning vector pPUSCSF, pPUSCSFGFP were successfully constructed. Recombinant hGM-CSF Lactococcus lactis secretion expressive vector pTRCSF and pTRCSFGFP were successfully constructed.4. Recombinant hGM-CSF Lactococcus lactis LL-CSF and LL-CSFGFP were successfully constructed.5. Recombinant hGM-CSF Lactococcus lactis LL-CSF could secretorily express hGM-CSF protein with bioactivity.6 Recombinant Lactococcus lactis we constructed was able to located in colon and express secretorily heterologous protein.7. Cytokines IL-6, IL-18, IL-12p35, IL-12p40, IFN-γhave taken part in the pathological mechanism of TNBS-induced colitis in rats, TNBS-induced colitis in rats is Th1-associated colitis which is similar to human CD, LL-CSF has obvious protective effect on TNBS-induced colitis in rats which may be related to downregulating cytokines IL-6, IL-12p35, IFN-γmRNA.
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