LL37 Microspheres Loaded on Activated Carbon-chitosan Hydrogel: Anti-bacterial and Anti-toxin Wound Dressing for Chronic Wound Infections.

Antimicrobial peptide LL37 is a promising antibacterial candidate due to its potent antimicrobial activity with no known bacterial resistance. However, intrinsically LL37 is susceptible to degradation in wound fluids limits its effectiveness. Bacterial toxins which are released after cell lysis are found to hinder wound healing. To address these challenges, encapsulating LL37 in microspheres (MS) and loading the MS onto activated carbon (AC)-chitosan (CS) hydrogel. This advanced wound dressing not only protects LL37 from degradation but also targets bacterial toxins, aiding in the healing of chronic wound infections. First, LL37 MS and LL37-AC-CS hydrogel were prepared and characterised in terms of physicochemical properties, drug release, and peptide-polymer compatibility. Antibacterial and antibiofilm activity, bacterial toxin elimination, cell migration, and cell cytotoxicity activities were investigated. LL37-AC-CS hydrogel was effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. LL37-AC-CS hydrogel bound more endotoxin than AC with CS hydrogel alone. The hydrogel also induced cell migration after 72 h and showed no cytotoxicity towards NHDF after 72 h of treatment. In conclusion, the LL37-AC-CS hydrogel was shown to be a stable, non-toxic advanced wound dressing method with enhanced antimicrobial and antitoxin activity, and it can potentially be applied to chronic wound infections to accelerate wound healing.

Associations of Serum Antimicrobial Peptide LL-37 with Longitudinal Cognitive Decline and Neurodegeneration Among Older Adults with Memory Complaints.

BACKGROUND: A potential role of the antimicrobial peptide LL-37, which is upregulated after infection, in the pathogenesis of Alzheimer's disease (AD) was identified. However, the clinical relevance of LL-37 in AD is not clear yet. OBJECTIVE: This study aims to investigate the association of circulating LL-37 with longitudinal cognitive decline and neurodegeneration among older adults with memory complaints. METHODS: This cohort study recruited 357 older adults with memory complaints. Participants were followed-up for two years and the cognitive functions were assessed using the Mini-Mental State Examination (MMSE). Serum LL-37, pTau181, and tTau levels were determined at baseline. Associations of baseline LL-37 with longitudinal cognitive decline and change of neurodegenerative biomarkers were analyzed. RESULTS: No difference was found in the slope of longitudinal cognitive decline during follow-up between the low and high LL-37 group, adjusting for age, sex, education, body mass index, APOE ɛ4 carrier status, comorbidities, and baseline MMSE scores (difference in slope: 0.226, 95% CI: -0.169 to 0.621). Higher LL-37 levels were associated with longitudinal cognitive decline, as indicated by a decrease of MMSE scores of 3 points or above during follow-up (OR = 2.11, 95% CI: 1.32 to 3.38). The high LL-37 group had larger slopes of the increase in neurofilament light (difference in slope: 3.759, 95% CI: 2.367 to 5.152) and pTau181 (difference in slope: 0.325, 95% CI: 0.151 to 0.499) than the low LL-37 group. CONCLUSION: These findings support an association of the antimicrobial peptide LL-37 with AD from a clinical perspective.

Antimicrobial Peptides Can Generate Tolerance by Lag and Interfere with Antimicrobial Therapy.

Antimicrobial peptides (AMPs) are widely distributed molecules secreted mostly by cells of the innate immune system to prevent bacterial proliferation at the site of infection. As with classic antibiotics, continued treatment with AMPs can create resistance in bacteria. However, whether AMPs can generate tolerance as an intermediate stage towards resistance is not known. Here, we show that the treatment of Escherichia coli with different AMPs induces tolerance by lag, particularly for those peptides that have internal targets. This tolerance can be detected as different morphological and physiological changes, which depend on the type of peptide molecule the bacterium has been exposed to. In addition, we show that AMP tolerance can also affect antibiotic treatment. The genomic sequencing of AMP-tolerant strains shows that different mutations alter membrane composition, DNA replication, and translation. Some of these mutations have also been observed in antibiotic-resistant strains, suggesting that AMP tolerance could be a relevant step in the development of antibiotic resistance. Monitoring AMP tolerance is relevant vis-á-vis the eventual therapeutic use of AMPs and because cross-tolerance might favor the emergence of resistance against conventional antibiotic treatments.

The effects of LL-37 on virulence factors related to the quorum sensing system of Pseudomonas aeruginosa.

Background: Antimicrobial peptides (AMPs) have shown promise in the treatment of multi-resistant pathogens. It was therefore of interest to analyze the effects of the AMP LL-37 on the regulation of several virulence factors related to the quorum sensing (QS) system of Pseudomonas aeruginosa (P. aeruginosa) in vitro. Methods: The minimum inhibitory concentration (MIC) was evaluated by the micro broth dilution method. The expression of QS-related and QS-regulated virulence factor genes was also evaluated. Exotoxin A activity was measured with the nicotinamide adenine dinucleotide (NAD) (Coenzyme I) method; Elastase activity was detected with the elastin-Congo red (ECR) method; Pyocyanin detection was performed using the chloroform extraction method. The effects of LL-37 were assessed by measuring the expression changes of the virulence protein-encoding genes of the strains with quantitative polymerase chain reaction (PCR). Results: The MIC of LL-37 against both P. aeruginosa reference strain (ATCC 15692 PAO1) and PA-ΔlasI/rhII was therefore determined to be 256 µg/mL. LL-37 at sub-minimum inhibitory concentrations (sub-MICs) had no significant effects on P. aeruginosa bacterial growth (P>0.05), but significantly downregulated the expression of all 3 virulence factors. Conclusions: Interestingly, this effect appeared to be dose-related. These findings suggest that LL-37 could be a potential candidate for QS inhibition against bacterial infection and may have significant clinical potential in the treatment of P. aeruginosa biofilms.

Codelivery of 1α,25-Dihydroxyvitamin D3 and CYP24A1 Inhibitor VID400 by Nanofiber Dressings Promotes Endogenous Antimicrobial Peptide LL-37 Induction.

Surgical site infections represent a significant clinical problem. Herein, we report a nanofiber dressing for topical codelivery of immunomodulating compounds including 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and VID400, a CYP24A1 inhibitor in a sustained manner, for inducing the expression of the endogenous cathelicidin antimicrobial peptide (CAMP) gene encoding the hCAP18 protein, which is processed into the LL-37 peptide. Nanofiber wound dressings with coencapsulation of 1,25(OH)2D3 and VID400 were generated by electrospinning. Both 1,25(OH)2D3 and VID400 were coencapsulated into nanofibers with loading efficiencies higher than 90% and exhibited a prolonged release from nanofiber membranes longer than 28 days. Incubation with 1,25(OH)2D3/VID400-coencapsulated poly(ϵ-caprolactone) nanofiber membranes greatly induced the hCAP18/LL-37 gene expression in monocytes, neutrophils, and keratinocytes in vitro. Moreover, the administration of 1,25(OH)2D3/VID400-coencapsulated nanofiber membranes dramatically promoted the hCAP18/LL-37 expression in dermal wounds created in both human CAMP transgenic mice and human skin tissues. The 1,25(OH)2D3- and VID400-coencapsulated nanofiber dressings enhanced innate immunity via the more effective induction of antimicrobial peptide than the free drug alone or 1,25(OH)2D3-loaded nanofibers. Together, 1,25(OH)2D3/VID400-embedded nanofiber dressings presented in this study show potential in preventing surgical site infections.

Evaluation of the T-SPOT.TB test, oxidation-related factors, and antimicrobial peptide LL-37 in the diagnosis of pulmonary tuberculosis with type 2 diabetes.

OBJECTIVE: To investigate the diagnostic value of the T cell spot (T-SPOT.TB) test, oxidation-related factors (ORF), and antimicrobial peptide LL-37 in patients with pulmonary tuberculosis (PTB) with type 2 diabetes. METHODS: A total of 560 patients with PTB admitted to our hospital from January 2019 to April 2021 were retrospectively included in this study. Of these, 232 patients with PTB and type 2 diabetes were assigned to the combined group, and 328 patients without complications were assigned to the PTB group. RESULTS: Areas under the curve (AUCs) for the number of spot-forming cells in CFP10 and ESAT-6 test panels detecting PTB with type 2 diabetes were 0.892 (95% confidence interval [CI] 0.831-0.921) and 0.893 (95% CI 0.841-0.935), respectively. CFP10 combined with ESAT-6 had the highest diagnostic value, with sensitivity and specificity levels and an AUC of 87.73%, 88.93%, and 0.942 (95% CI 0.907-0.967), respectively. The levels of total antioxidant capacity, superoxide dismutase, and catalase in the combined group were lower than in PTB and control groups. CONCLUSION: The combination of T-SPOT.TB, ORF, and LL-37 in the diagnosis of pulmonary tuberculosis with type 2 diabetes mellitus has a high diagnostic value and clinical application value.

Antimicrobial peptide LL-37 ameliorates a murine sepsis model via the induction of microvesicle release from neutrophils.

Sepsis is a life-threatening disease caused by systemic dys-regulated inflammatory response to infection. We previously revealed that LL-37, a human cathelicidin antimicrobial peptide, improves the survival of cecal ligation and puncture septic mice. Ectosomes, microvesicles released from neutrophils, are reported to be elevated in sepsis survivors; however, the functions of ectosomes in sepsis remain largely unknown. Therefore, we herein elucidated the protective action of LL-37 on sepsis, by focusing on LL-37-induced ectosome release in a cecal ligation and puncture model. The results demonstrated the enhancement of ectosome levels by LL-37 administration, accompanied by a reduction of bacterial load. Importantly, ectosomes isolated from LL-37-injected cecal ligation and puncture mice contained higher amounts of antimicrobial proteins/peptides and exhibited higher antibacterial activity, compared with those from PBS-injected cecal ligation and puncture mice, suggesting that LL-37 induces the release of ectosomes with antibacterial potential in vivo. Actually, LL-37 stimulated mouse bone-marrow neutrophils to release ectosomes ex vivo, and the LL-37-induced ectosomes possessed antibacterial potential. Furthermore, administration of LL-37-induced ectosomes reduced the bacterial load and improved the survival of cecal ligation and puncture mice. Together these observations suggest LL-37 induces the release of antimicrobial ectosomes in cecal ligation and puncture mice, thereby reducing the bacterial load and protecting mice from lethal septic conditions.

Inhibitory effect of antimicrobial peptide LL-37 on tumor growth of mice with colon cancer and its mechanism / 吉林大学学报(医学版)

Objective: To investigate the effects of antimicrobial peptide LL-37 on the tumor growth and apoptosis of the mice with colon cancer, and to elucidate the possible molecular mechanism of its anti-tumor effect. Methods: The LL-37 over-expression colon cancer HT-29 cells were constructed, and the expression levels of LL-37 mRNA and protein in the HT-29 cells were detected by qRT-PCR and Western blotting methods. A total of 30 BALB/c mice were randomly divided into control group (given the uninfected HT-29 cells)' empty vector group (given the HT-29 cells infected with empty plasmid), LL-37 over-expression group (given the HT-29 cells infected with LL-37 over-expression vector), AMPK inhibitor group [given the HT-29 cells infected with empty vector, and then injected with 2 mg • kg Dorsomorphin (Dor) in the tail vein

Evaluation of plasma antimicrobial peptide LL-37 and nuclear factor-kappaB levels in stable chronic obstructive pulmonary disease.

BACKGROUND: Antimicrobial peptides are effectors of host defence against infection and inflammation and can encourage wound repair. OBJECTIVES: The objectives of this study were to investigate the plasma antimicrobial peptide LL-37 and nuclear factor-kappaB (NF-κB) levels in patients with stable COPD compared with a control group and to highlight their importance in immune inflammation. METHODS: One hundred and thirty-eight stable COPD patients and 33 control subjects were enrolled in the study. The COPD patients were classified into four groups based on FEV1 (groups I-IV) and also divided into "low-risk and high-risk" groups (groups A-B [low risk], C-D [high risk]). RESULTS: Plasma LL-37 levels were significantly lower while plasma NF-κB levels of the COPD patients were significantly higher than those of the control subjects (P<0.001, both). LL-37 levels were significantly lower in group IV than in groups I, II, and III (P<0.01, all). NF-κB levels were significantly higher in groups III and IV than in groups I and II (P<0.05, both). There was a positive correlation between FEV1 and FEV1/FVC in all COPD patients (r=0.742, P<0.001) and in group D (r=0.741, P<0.001). Furthermore, there was an inverse correlation between LL-37 and NF-κB in both the groups C (r=-0.566, P<0.001) and D (r=-0.694, P<0.001) and group C+D combined (r=-0.593, P<0.001). Furthermore, in group C, LL-37 and FEV1 were positively correlated (r=0.633, P<0.001). CONCLUSION: Our study indicated that plasma LL-37 and NF-κB may play an important role in chronic immune inflammation. Decreased LL-37 levels may be particularly high risk for patients in stage IV disease. The role of LL-37 as a target for treatment of the immune system and COPD must be widely evaluated.

Astragalus polysaccharides exerts anti-infective activity by inducing human cathelicidin antimicrobial peptide LL-37 in respiratory epithelial cells.

Astragalus polysaccharides (APS), one of the major active components in Astragalus membranaceus, is an effective immunomodulator used in the treatment of immunological diseases in China. However, the anti-infective action and mechanism of APS is not fully known. In the present study, we found that APS induced the expression of human cathelicidin antimicrobial peptide LL-37, a key host anti-infective molecule, in both mRNA and protein levels in respiratory epithelial cells HBE16 and A549. Furthermore, the lysate and supernatant from APS-treated HBE16 cells both exhibited an obvious antibacterial action, which was partially neutralizated by LL-37 monoclonal antibody. In addition, APS also significantly elevated the phosphorylation of p38 MAPK and JNK and caused the degradation of IκBα. Specific inhibitors of p38 MAPK, JNK, or NF-κB obviously abolished APS-induced LL-37 synthesis and antibacterial activity, respectively. Taken together, our results confirmed the enhancement of APS on LL-37 induction and antibacterial action in respiratory epithelial cells, which may be attributed to activation of p38 MAPK/JNK and NF-κB pathways. Furthermore, these results also supported the clinical application of APS in the treatment of infectious diseases.

Changes and clinical significance of antimicrobial peptide LL-37 and CRP in patients with Pseudomonas aeruginosa positive sputum culture / 国际检验医学杂志

Objective To investigate the changes of antimicrobial peptide LL-37 and C-reactive protein (CRP ) in the patients with Pseudomonas aeruginosa positive sputum culture and their mutual relation . Methods Fifty cases of Pseudomonas aeruginosa positive sputum culture and 27 cases undergoing physical ex-amination in the Guangdong Provincial Hospital of Chinese Medicine from September 2016 to May 2017 were selected as the research subjects .The level of antimicrobial peptide LL-37 was detected by double antibody sandwich ELISA and the CRP level was detected by immunoturbidimetry .Results The levels of antimicrobial peptide LL-37 and CRP in the Pseudomonas aeruginosa positive sputum culture group were significantly high-er than those in the healthy control group ,the difference was statistically significant (P< 0 .05) ,and they showed the positive correlation (r=0 .411 ,P<0 .05) .Conclusion In positive sputum culture of Pseudomonas aeruginosa ,antimicrobial peptide LL-37 and CRP levels are increased ,which has a certain clinical application value for the early diagnosis of infection .

Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria.

UNLABELLED: Electronic (e)-cigarette use is rapidly rising, with 20 % of Americans ages 25-44 now using these drug delivery devices. E-cigarette users expose their airways, cells of host defense, and colonizing bacteria to e-cigarette vapor (EV). Here, we report that exposure of human epithelial cells at the air-liquid interface to fresh EV (vaped from an e-cigarette device) resulted in dose-dependent cell death. After exposure to EV, cells of host defense-epithelial cells, alveolar macrophages, and neutrophils-had reduced antimicrobial activity against Staphylococcus aureus (SA). Mouse inhalation of EV for 1 h daily for 4 weeks led to alterations in inflammatory markers within the airways and elevation of an acute phase reactant in serum. Upon exposure to e-cigarette vapor extract (EVE), airway colonizer SA had increased biofilm formation, adherence and invasion of epithelial cells, resistance to human antimicrobial peptide LL-37, and up-regulation of virulence genes. EVE-exposed SA were more virulent in a mouse model of pneumonia. These data suggest that e-cigarettes may be toxic to airway cells, suppress host defenses, and promote inflammation over time, while also promoting virulence of colonizing bacteria. KEY MESSAGE: Acute exposure to e-cigarette vapor (EV) is cytotoxic to airway cells in vitro. Acute exposure to EV decreases macrophage and neutrophil antimicrobial function. Inhalation of EV alters immunomodulating cytokines in the airways of mice. Inhalation of EV leads to increased markers of inflammation in BAL and serum. Staphylococcus aureus become more virulent when exposed to EV.

Does smoking affect gingival crevicular fluid LL-37 levels following non-surgical periodontal treatment in chronic periodontitis?

OBJECTIVE: LL-37 contributes to maintaining the balance between health and disease. Smoking is a risk factor for periodontitis that impairs neutrophil functions. The aim of the present study was to comparatively evaluate gingival crevicular fluid (GCF) LL-37 levels in smoker and non-smoker chronic periodontitis (CP) patients and controls, as well as the effect of non-surgical periodontal treatment on GCF LL-37 levels. DESIGN: Thirty-one CP patients (16 smokers, 15 non-smokers) and thirty-one controls (16 smokers, 15 non-smokers) were included in the study. CP patients received non-surgical treatment. GCF LL-37 levels and periodontal parameters were assessed at baseline, 1 and 3 months after completion of non-surgical periodontal treatment. GCF LL-37 levels were analyzed by ELISA. RESULTS: No significant difference was observed in GCF LL-37 levels between smoker and non-smoker controls (p>0.05). Smoker CP group had significantly lower GCF LL-37 level than non-smoker CP group at baseline (p<0.05). GCF LL-37 levels significantly decreased in non-smoker CP group at first week, 1 and 3 months after completion of non-surgical periodontal treatment (p<0.05) although no significant decrease in GCF LL-37 levels was observed in smoker CP group (p>0.05). Periodontal parameters were correlated with GCF LL-37 levels in non-smoker CP group (p<0.05), but not in smoker CP group (p>0.05). CONCLUSIONS: GCF LL-37 levels do not seem to be affected from smoking in periodontal health. However, smoking might have a suppressive effect on GCF LL-37 levels in CP. Non-surgical treatment is effective in decreasing GCF LL-37 levels in non-smoker CP patients but not in smokers with CP.

Antimicrobial peptide LL-37 along with peptidoglycan drive monocyte polarization toward CD14(high)CD16(+) subset and may play a crucial role in the pathogenesis of psoriasis guttata.

The human cathelicidin LL-37 peptide is overexpressed in psoriasis and has been demonstrated to be a multifunctional modulator of innate immune response elements, including monocytes. Monocytes, categorized into three populations based on the cell surface expression of CD14 and CD16, are activated in psoriasis guttate and are commonly triggered by streptococcal infections. Peptidoglycan (PGN) is a major cell-wall component of streptococcus, and an increasing number of PGN-containing cells have been detected in psoriasis. Since there are independent reports of both PGN and LL-37 influencing monocytes, we tried to evaluate the effect of human LL-37 on PGN-induced monocyte activity and differentiation and subsequently studied their correlation with the pathogenesis of psoriasis guttate. The results revealed that monocytes from the peripheral blood of healthy individuals resulted in their polarization toward the CD14(high)CD16(+) subset, when cultured with PGN in the presence of the LL-37 peptide. This peptide further induced PGN-driven differentiated monocytes into immature dendritic cells (iDC), as evident by the increased expression of CD1a, CD86, and HLA-DR markers, resulting in the induction of T cell proliferation and Th17 polarization. Furthermore, our data suggested that psoriasis guttata patients have significantly higher percentages of CD14(high)CD16(+) monocytes as well as circulating levels of LL-37, soluble form of triggering receptor expressed on myeloid cells (sTREM-1) levels, and anti-streptolysin O (ASO) levels, as compared to healthy controls. Psoriasis guttata patients also showed a positive correlation between the percentage of CD14(high)CD16(+) monocytes and the serum levels of sTREM-1 as well as the Psoriasis Area and Severity Index (PASI) scores. Therefore, we concluded that LL-37 in synergy with PGN directs monocyte polarization and differentiation into a proinflammatory phenotype, which might play a crucial role in the pathogenesis of psoriasis.

Salivary concentration of the antimicrobial peptide LL-37 in patients with oral lichen planus.

BACKGROUND: The antimicrobial peptide LL-37 is a significant molecule of innate immunity and recent studies indicate that it plays an important role in maintaining oral health. Yet limited knowledge exists on its role in oral diseases and oral lichen planus (OLP) in particular. OBJECTIVE: The study aimed to examine: 1) the salivary concentration of LL-37 in patients with OLP and healthy subjects, 2) the relation between the type (reticular or erosive) and size of OLP lesions and LL-37 concentration, and 3) the effect of the therapeutic modalities on LL-37 levels. DESIGN: The salivary peptide concentration in samples from 20 patients and 30 healthy subjects at the same age range was determined by ELISA. RESULTS: Despite the wide variation in peptide concentration found in both groups, the healthy subjects exhibited significantly lower levels than patients. Patients with the erosive form had significantly higher peptide concentrations than patients with the reticular form. Systemic treatment with corticosteroids resulted in a significant decrease of the salivary peptide concentration, while other treatment modalities, such as administration of vitamins A and E or local application of corticosteroids had no effect. Improved clinical appearance of the lesions was followed by a decrease in the salivary LL-37 level. CONCLUSIONS: Salivary concentration of LL-37 correlates to the manifestation of mucosa lesions in OLP patients, the highest levels being observed in the most severe cases. This increase in peptide levels may protect against lesion infection and promote a quick wound healing.

Modification of 5′-UTR sequences of pPIC9 increases expression of antimicrobial peptide LL-37 / 第二军医大学学报

Objective: To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris. Methods: The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was transformed into E. coli DH5α to construct a modified eukaryotic vector pPIC9-EDIT. After PCR and sequencing, pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast, the product was then transformed into E. coli DH5α to construct the recombinant expression vector pPIC9-EDIT-LL-37, the latter was transformed into P. pastoris GS115 by spheroplasting and the insert was confirmed by PCR. The bacteriolytic activity to E. coli. DH5α was analyzed to screen the highest expressing strain and to determine the best inducing time and concentration of methanol. The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting. The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-EDIT-LL-37 were compared, and the changes of LL-37 protein expression were determined before and after modification. Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed. Expression of LL-37 gene was confirmed by PCR in P. pastoris after pPIC9-EDIT-LL-37 transformation. The highest expressing strain was identified; the best inducing time was 72 h and the best concentration of methanol was 0.5%. Tricine-SDS-PAGE and Western blotting analysis showed that the expression product was LL-37. The expression level of LL-37 protein increased by 35 times after modification. Conclusion: Modification of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P. pastoris; it is worth to be used in the research of other heterogenous protein.

Modification of 5′-UTR sequences of pPIC9 increases expression of antimicrobial peptide LL-37 / 第二军医大学学报

Objective:To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris.Methods:The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was trans- formed into E.coli DH5?to construct a modified eukaryotic vector pPIC9-EDIT.After PCR and sequencing,pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast,the product was then transformed into E.coli DH5?to con- struct the recombinant expression vector pPIC9-EDIT-LL-37,the latter was transformed into P.pastoris GS115 by spheroplas- ting and the insert was confirmed by PCR.The bacteriolytic activity to E.coli.DH5?was analyzed to screen the highest ex- pressing strain and to determine the best inducing time and concentration of methanol.The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting.The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-ED- IT-LL-37 were compared,and the changes of LL-37 protein expression were determined before and after modification.Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed.Expression of LL-37 gene was confirmed by PCR in P.pastoris after pPIC9-EDIT-LL-37 transformation.The highest expressing strain was identified;the best inducing time was 72 h and the best concentration of methanol was 0.5%.Tricine-SDS-PAGE and Western blotting analysis showed that the ex- pression product was LL-37.The expression level of LL-37 protein increased by 35 times after modification.Conclusion:Modifi- cation of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P.pastoris;it is worth to be used in the research of other heterogenous protein.