Antigenicity of tissues and organs from GGTA1/CMAH/β4GalNT2 triple gene knockout pigs

Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigens[Gal,Neu5 Gc,and Sd(a)]present on pig cells and tissues.Triple gene(GGTAI,CMAH,and β4 GalNT2)knockout(TKO)pigs were produced in our laboratory by CRISPR-Cas9 targeting.To investigate the antigenicity reduction in the TKO pigs,the expression levels of these three xenoantigens in the cornea,heart,liver,spleen,lung,kidney,and pancreas tissues were examined.The level of human IgG/IgM binding to those tissues was also investigated,with wildtype pig tissues as control.The results showed that aGal,Neu5 Gc,and Sd(a)were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs.Compared to wildtype pigs,the liver,spleen,and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding,whereas corneas,heart,lung,and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding.These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.

Effect of extrusion on the structure and antigenicity of soybean β-conglycinin

β-Conglycinin,the main component of 7S globulin in soybean protein,is also a key soybean antigen protein that causes allergic reactions.Extrusion technologies have received considerable attention as amethod for modifying soybean protein allergens.This study investigated the changes inβ-conglycinin structure and antigenicity upon extrusion.Isoelectric precipitation,ammoniumsulfate precipitation,and sepharose CL-6B gel filtration were used to isolate and purifyβ-conglycinin from soybean powder,and single-factor and orthogonal tests were used to study the effects of water content,extrusion temperature,screw rotation speed,and feeding speed on the antigenicity ofβ-conglycinin after extrusion.Fourier transforminfrared spectrometry(FTIR)was then employed to analyze the structure ofβ-conglycinin after extrusion under the optimal conditions determined by the orthogonal test.The results showed that extrusion significantly reduced the antigenicity ofβ-conglycinin(P<0.05),and the degree of influence of the factors studied may be ordered as extrusion temperature>feeding speed>screw rotation speed>water content.The optimal parameters were temperature at 130°C,screwrotation speed at 140 r/min,and feeding speed at 35 g/min.Under these conditions,the contents ofα-helix,β-pleated sheet,andβ-turn structures inβ-conglycinin were significantly reduced(P<0.05),while the contents of random coils were significantly increased(P<0.05).The peak absorption intensity of amides I,II,and III also decreased.Taken together,the findings suggest that extrusion could be an effective method for reducing the antigenicity ofβ-conglycinin.

Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Due to its beneficial health effects,the use of soybean protein has shown a continuous increase,but concerns regarding the allergenicity of soybean antigenic protein have also increased.This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybeanβ-conglycinin and the allergenicity of its hydrolysates.Alkaline protease of the strain was separated by precipitation method of organic solvents,and theβ-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column.Using the degree of hydrolysis(DH)and inhibition rate as evaluation indexes,the enzymatic hydrolysis parameters ofβ-conglycinin was optimized by single factor and L_(9)(3^(4))orthogonal tests,so as to explore the effect of the protease on the hydrolysis degree and the antigenicity ofβ-conglycinin hydrolysates.The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity.The purity ofβ-conglycinin was 84.8%.The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate,and the increase of protein concentration causedmonotone increasing and monotone decreasing of the inhibition rate and the DH,and the optimal protein concentration was 30 mg/mL.The optimization results showed that pH had the largest impacts on both DH and the inhibition rate,followed by enzyme dosage,hydrolysis temperature and hydrolysis time.Under the optimum hydrolysis conditions of protein concentration 30mg/mL,enzymedosage0.7%,hydrolysis time40min,temperature 55°C and pH8.5,the DH reached the highest of 76.28%,and the inhibition rate was the lowest of 27.03%,which was reduced greatly compared with that before optimization.These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity,making it possible to produce low-allergenicity soybean protein.

Enzymatic kinetics ofβ-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and analysis of antigenicity of hydrolyzed peptide

β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B.subtilis ACCC 01746.A dynamic model describing the hydrolysis ofβ-conglycinin was proposed using the initial substrate concentration,enzyme dosage(enzyme to substrate ratio)and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis.The hydrolysis of soybeanβ-conglycinin was carried out at 60 g/L protein concentration,0.6%enzyme dosage,55℃ and pH 8.5 to observe the peptides with anti-enzymatic activities.The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40,30,20,and 10 kDa,and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay.The results showed that the degree of hydrolysis(DH)ofβ-conglycinin decreased as theβ-conglycinin concentration(S0)increased,but increased with enzyme dosage(E0)increasing.Thus,the enzymatic hydrolysis ofβ-conglycinin followed the first-order kinetics model.The hydrolysis rate(V)was(527.89C_(E0)-2.5533C_(S0))exp(-0.022DH),the DH-hydrolysis time was 45.454ln[1+(11.614C_(E0)/C_(S0)-0.0562)t],and the correlated kinetic constants k2 and kd were 527.89 min^(−1)and 8.6126 min^(−1),respectively.The hydrolysis behavior ofβ-conglycinin varied considerably among theα',α,andβsubunits.Faster hydrolysis rates were observed for theα'andαsubunits compared to theβsubunit.The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8-40.1 kDa,and the antigenicity of the peptides with smaller molecular weight was reduced,but not removed completely.However,the alkaline protease from the strain appeared to effectively reduce the allergenicity ofβ-conglycinin.Therefore,it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis.Additionally,the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.

The Secondary Structure of Heated Whey Protein andIts Hydrolysates Antigenicity

Fourier transform infrared spectroscopy(FTIR) and circular dichroism(CD) were used to investigate the conformational changes of heated whey protein(WP) and the corresponding changes in the hydrolysates immunoreactivity were determined by competitive enzyme-linked immunosorbent assay(ELISA).Results showed that the contents of α-helix and β-sheet of WP did not decrease much under mild heating conditions and the antigenicity was relatively high;when the heating intensity increased(70 ℃ for 25 min or 75 ℃ for 20 min),the content of α-helix and β-sheet decreased to the minimum,so was the antigenicity;However,when the WP was heated at even higher temperature and for a longer time,the β-sheet associated with protein aggregation begun to increase and the antigenicity increased correspondingly.It was concluded that the conformations of heated WP and the antigenicity of its hydrolysates are related and the optimum structure for decreasing the hydrolysates antigeniity is the least content of α-helix and β-sheet.Establishing the relationship between the WP secondary structure and WP hydrolysates antigenicity is significant to supply the reference for antigenicity reduction by enzymolysis.

Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS 1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction （RT-PCR） and cloned into a prokaryotic expression vector, pET-28a（＋）, and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay （ELISA） was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

EXPERIMENTAL STUDY OF INFLUENCE OF LIQUID NITROGEN PRESERVATION ON ANTIGENICITY OF HUMAN HOMOGRAFT AORTIC VALVE

To illustrate the effect of liquid nitrogen preservation on antigenicity of human homograft aortic valve (HAV), human aortic valve tissue were cocultured together with peripheral blood mononuclear cells (PBMCs). Following detections were done to show the difference of antigenicity of HAV indirectly at different period after being preserved in liquid nitrogen: ① 3H TdR incorporation (cpm) was observed after tissue cell cocultured and stimulation index (SI) was calculated;② Density of IL 2 in medium was measured by MTT means. The results indicated that antigenicity of fresh HAV was the strongest; with the prolong of being preserved, antigenicity decreased gradually. It decreased significantly within first 48 h preserved at 4℃, then decreased significantly after preserved in liquid nitrogen for 24 h. 2 weeks later, antigenicity decreased to the lowest level.

The study of differences of antigenicity in rabbit intervertebral disc

Objective To reveal and compare the immunogenicity of glycoprotein from nucleus pulposus and collagen from anulus fibrosus in rabbit intervertebral disc.Methods According to the similarity principle of genome biology and the amount

Prediction, Synthesis and Antigenicity of the Antigenic Peptides of 26kDa Glutathione S-Transferasc of Schistosoma Japonicum (Sj26)

Six antigenic peptides of 26 kDa glutathione S-transferase of Schistosoma japonicum(Sj26) have been predicted according to their hydrophilicity, flexibility. accessibility. chargedistribution and β -turn in the secondary structure by the determination of its primary structure andsynthesized by solid phase method. All of them showed antigenicity with anti-schistosomajaponicum immunoglobulin polyclonal antibody, anti-Sj-lgG PcAb by Dot-ELISA. Three of themshowed good antigenicity. They would serve as candidates of synthetic anti-schistosomal vaccine.

The development of a recombinant hybrid protein of Plasmodium falciparum and analysis of its antigenicity and protectivity

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Removal of intrahepatic bile duct stone could reduce the risk of cholangiocarcinoma: A single-center retrospective study in South Korea

BACKGROUND Intrahepatic duct(IHD)stones are among the most important risk factors for cholangiocarcinoma(CCC).Approximately 10%of patients with IHD stones develop CCC;however,there are limited studies regarding the effect of IHD stone removal on CCC development.AIM To investigate the association between IHD stone removal and CCC development.METHODS We retrospectively analyzed 397 patients with IHD stones at a tertiary referral center between January 2011 and December 2020.RESULTS CCC occurred in 36 of the 397 enrolled patients.In univariate analysis,chronic hepatitis B infection(11.1%vs 3.0%,P=0.03),carbohydrate antigen 19-9(CA19-9,176.00 vs 11.96 II/mL,P=0.010),stone located in left or both lobes(86.1%vs 70.1%,P=0.042),focal atrophy(52.8%vs 26.9%,P=0.001),duct stricture(47.2%vs 24.9%,P=0.004),and removal status of IHD stone(33.3%vs 63.2%,P<0.001)were significantly different between IHD stone patients with and without CCC.In the multivariate analysis,CA19-9>upper normal limit,carcinoembryonic antigen>upper normal limit,stones located in the left or both lobes,focal atrophy,and complete removal of IHD stones without recurrence were independent factors influencing CCC development.However,the type of removal method was not associated with CCC risk.CONCLUSION Complete removal of IHD stones without recurrence could reduce CCC risk.

The Structural Characterization and Antigenicity of the S Protein of SARS-CoV

The corona-like spikes or peplomers on the surface of the virion under electronic microscope are the most striking features of coronaviruses. The S (spike) protein is the largest structural protein, with 1,255 amino acids, in the viral genome. Its structure can be divided into three regions: a long N-terminal region in the exterior, a characteristic transmembrane (TM) region, and a short C-terminus in the interior of a virion. We detected fifteen substitutions of nucleotides by comparisons with the seventeen published SARS-CoV genome sequences, eight (53.3%) of which are non-synonymous mutations leading to amino acid alternations with predicted physiochemical changes. The possible antigenic determinants of the S protein are predicted, and the result is confirmed by ELISA (enzyme-linked immunosorbent assay) with synthesized peptides. Another profound finding is that three disulfide bonds are defined at the C-terminus with the N-terminus of the E (envelope) protein, based on the typical sequence and positions, thus establishing the structural connection with these two important structural proteins, if confirmed. Phyloge-netic analysis reveals several conserved regions that might be potent drug targets.

The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Parallel profiling of antigenicity alteration and immune escape of SARS-CoV-2 Omicron and other variants

SARS-CoV-2 variants have evolved a variety of critical mutations,leading to antigenicity changes and immune escape.The recent emerging SARS-CoV-2 Omicron variant attracted global attention due to its significant resistance to current antibody therapies and vaccines.

Evaluation of the infectivity,gene and antigenicity persistence of rotaviruses by free chlorine disinfection

The effects of free chlorine disinfection of tap water and wastewater effluents on the infectivity, gene integrity and surface antigens of rotaviruses were evaluated by a bench-scale chlorine disinfection experiments. Plaque assays, integrated cell culture-quantitative RT- PCR （ICC-RT-qPCR）, RT-qPCR, and enzyme-linked immunosorbent assays （ELISA）, respectively, were used to assess the influence of the disinfectant on virus infectivity as well as genetic and antigenic integrity of simian rotavirus SA11 as a surrogate for human rotaviruses. The ICC-RT-qPCR was able to detect rotaviruses survival from chlorine disinfection at chlorine dose up to 20 mg/L （60 min contact）, which suggested a required chlorine dose of 5 folds （from 1 to 5 mg/L） higher than that indicated by the plaque assay to achieve 1.8 log10 reductions in tap water with 60 rain exposing. The VP7 gene was more resistant than the infectivity and existed at chlorine dose up to 20 mg/L （60 min contact）, while the antigencity was undetectable with chlorine dose more than 5 mg/L （60 min contact）. The water quality also impacted the inactivation efficiencies, and rotaviruses have a relatively higher resistant in secondary effluents than in the tap water under the same chlorine disinfection treatments. This study indicated that rotaviruses have a higher infectivity, gene and antigencity resistance to chlorine than that previously indicated by plaque assay only, which seemed to underestimate the resistance of rotaviruses to chlorine and the risk of rotaviruses in environments. Present results also suggested that re-evaluation of resistance of other waterborne viruses after disinfections by more sensitive infectivity detection method （such as ICC-RT-qPCR） may be necessary, to determine the adequate disinfectant doses required for the inactivation of waterborne viruses.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment.To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine,the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared.The neutralization activities of Alpha,Beta and Gamma variants’spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant,which were constructed using pseudotyped viruses.Meanwhile,the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants.Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011–2019.Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds,whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10–100-fold.Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results.This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

SDS-PAGE PROTEIN PATTERN AND ITS ANTIGENICITY ANALYSIS OF DIFFERENT ISOLATES OF SCHISTOSOMA JAPONICUM IN CHINA

Homogenates prepared from S. japonicum adult worms ofdifferent isolates from Anhui, Hubei, Guangxi, Yunnan andSichuan Provinces were analyzed by SDS-PAGE and enzymelinked immunoelectrotransfer blot (EITB) tested with rabbitanti-snails antibody. The results of SDS-PAGE indicated thatwith silver staining both male and female worms of Guangxiisolate showed some definite differences in their protein profile,namely, absence of one band between 50-75 kDa in maleworms and marked reduction in quantity of > 110 and 30 kDabands in female worms. There was no obvious differenceamong other isolates both in male and female worms. TheEITB patterns were similar in S. japonicum of Anhui andHubei, and it was also the case with isolates from Yunnan andSichuau, except that Yuunan female worms had a distinct band

Serum tumor markers expression(CA199,CA242,and CEA)and its clinical implications in type 2 diabetes mellitus

BACKGROUND Glucose and lipid metabolic disorder in patients with type 2 diabetes mellitus(T2DM)is associated with the levels of serum tumor markers of the digestive tract,such as cancer antigen(CA)199.Therefore,tumor markers in T2DM are important.AIM To evaluate the expression of serum tumor markers[CA199,CA242,and carcinoembryonic antigen(CEA)]and the clinical implications of the expression in T2DM.METHODS For this observational study conducted at Hefei BOE Hospital,China,we enrolled 82 patients with first-onset T2DM and 51 controls between April 2019 and December 2020.Levels of fasting blood glucose(FBG),tumor markers(CA199,CEA,and CA242),glycosylated hemoglobin(HbA1c),etc.were measured and group index levels were compared.Moreover,FBG and HbA1c levels were correlated with tumor marker levels.Tumor markers were tested for diagnostic accuracy in patients with>9%HbA1c using the receiver operating curve(ROC)curve.RESULTS The T2DM group had high serum FBG,HbA1c,CA199,and CEA levels(P<0.05).A comparative analysis of the two groups based on HbA1c levels(Group A:HbA1c≤9%;Group B:HbA1c>9%)revealed significant differences in CEA and CA199 levels(P<0.05).The areas under the ROC curve for CEA and CA199 were 0.853 and 0.809,respectively.CA199,CEA,and CA242 levels positively correlated with HbA1c(r=0.308,0.426,and 0.551,respectively)and FBG levels(r=0.236,0.231,and 0.298,respectively).CONCLUSION As compared to controls,serum CEA and CA199 levels were higher in patients with T2DM.HbA1c and FBG levels correlated with CA199,CEA,and CA242 levels.Patients with poorly controlled blood sugar must be screened for tumor markers.

Antigen epitopes of animal coronaviruses:a mini-review

Coronaviruses are widespread in nature and can infect mammals and poultry,making them a public health concern.Globally,prevention and control of emerging and re-emerging animal coronaviruses is a great challenge.The mecha-nisms of virus-mediated immune responses have important implications for research on virus prevention and control.The antigenic epitope is a chemical group capable of stimulating the production of antibodies or sensitized lympho-cytes,playing an important role in antiviral immune responses.Thus,it can shed light on the development of diagnos-tic methods and novel vaccines.Here,we have reviewed advances in animal coronavirus antigenic epitope research,aiming to provide a reference for the prevention and control of animal and human coronaviruses.