CN112034181A - Application of lectin probe combination in identification of black-leaf monkey pregnancy based on urine protein glycoform - Google Patents
Application of lectin probe combination in identification of black-leaf monkey pregnancy based on urine protein glycoform Download PDFInfo
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Abstract
The invention provides an application of a lectin probe combination in identifying black-leaf monkey pregnancy based on a urine protein glycoform. The lectin probe combination is a combination of PHA-E, STL, PTL-I, PNA and SNA; the identification basis is as follows: if the sample detection result is that the expression of PHA-E, STL is up-regulated, and the expression of PTL-I, PNA and SNA is down-regulated, it indicates that the corresponding black-leaf monkeys are in the gestation period at the time of sampling. The method is used for identifying the pregnancy status of the black-leaf monkeys, is quick, simple, convenient and accurate, and has lower realization cost.
Description
Technical Field
The invention relates to a method for identifying whether black-leaf monkeys are in gestation period.
Background
In the aspect of rare wild animal protection, due to the damage and fragmentation of habitats, the population quantity is expanded by artificial participation modes such as captive breeding and scattered breeding, endangered species are protected on site or migrated to a proper environment, and the possibility is provided for the wild homing of the endangered species. Many endangered wildlife have poor self-reproductive abilities, such as clouded leopards, and male leopards can injure and even kill females when not familiar with each other. Therefore, attempts have been made to improve the reproduction rate of species by using assisted reproduction techniques such as artificial insemination and embryo transfer. These methods also offer the possibility of exchanging genetic material (sperm or embryos) between wild and captive animals, helping to maintain genetic diversity, and greatly aid in species management. However, the success rate of endangered species breeding is still low, people need to know the physiological characteristics of animals and grasp an effective pregnancy diagnosis means urgently. Techniques conventionally used for pregnancy diagnosis of animals include rectal examination, vaginal examination, diagnostic imaging, aphrodisiac, immunological diagnosis, hormone level measurement, and the like. However, the process of catching the animals can cause the animals to generate strong stress, the damage is larger, or the hormone level fluctuation is larger, so that a longer monitoring period is needed. There is a need for a more rapid and efficient method for breaking the traditional thought of pregnancy diagnosis.
The black-leaf monkey (Trachyphecus francoisi) is an animal of the family of the Piperaceae of the order Primates and is the most common one of the black-leaf monkeys in China, and the whole body including the hairs of hands and feet are black, and the back is longer and denser than the abdomen, so the black-leaf monkey is also called a black ape. The wild animals are distributed in Vietnam and Guizhou and Guangxi in China, mainly live in cliff, karst cave, canyon and other complex terrains in limestone hilly areas, are listed as seriously-endangered species by the International Union of Natural and Natural resource protection (IUCN) due to narrow distribution areas, severe habitat and rare population quantity, and are listed as the first-grade national key protection wild animals in China. The external shape of estrus is not obviously changed, the phenomenon of sexual skin redness and swelling is not obvious, the abdomen is hypertrophic, even in the later period of pregnancy, the obvious change from the posture is difficult to observe, and the sexual biliary disorder is small, the habitat topography is complex and the like, so that the human beings are difficult to access and study. At present, a simple and feasible method for diagnosing rare pregnancy as early as possible does not exist, pregnant mares cannot be protected in time, the phenomenon of abortion and stillbirth in the artificial breeding process is serious, the breeding rate is greatly influenced, and the development of artificial populations and the protection of transplanting are not facilitated.
Research has progressed over the years to show that due to the limited number of genes, modification of proteins is an important factor in regulating their function. Glycosylation is the most abundant and complex form of post-translational modification, has a significant impact on protein folding, partitioning, stability and activity, and is an important regulator of the properties of proteins and lipids present on cell surfaces. With the rapid development of glycomics, the search of individual pregnancy or even abnormal pregnancy biomarkers and the analysis of sugar chain differences between two sexes are new exploration directions which are generated with the new development of new technologies.
Glycomics can be defined as a comprehensive interpretation of all sugars (monosaccharides, oligosaccharides, polysaccharides and modifications thereof). Monosaccharides are the basic building blocks of sugar chains, and mammalian sugar chains are composed of only ten core monosaccharides. Under several enzymatic reaction conditions involving glycosidases and glycosyltransferases, the basic building blocks constitute diverse sugar chain structures. Eukaryotic protein glycosylation is usually achieved by glycosidic bonds with serine/threonine residues to form O-sugar chains, and aspartic acid residues to form N-sugar chains. More than 50% of mammalian proteins have sugar chain modifications, and glycoproteins present in all mammalian body fluids differ in the sugar chains that evolve between different species and phylogeny, which is an important marker of the immune system in distinguishing between self and non-self.
Urine is receiving more and more attention due to the characteristics of relatively simple protein components, convenient and noninvasive collection, very important clinical significance and the like. The lectin chip technology has the characteristics of high coverage rate, simple and convenient operation and quick experimental performance, can be designed according to the characteristic of identifying a specific polysaccharide structure of the lectin, extracts protein in urine, and simultaneously quantitatively analyzes N-sugar chains and O-sugar chains in a biological sample under the condition of not releasing the protein. Efforts such as sugar chain analysis of pregnant individuals with choriocarcinoma using lectin chips to find new cancer indicators and to detect differences in sugar chains on the surface of mammalian sperm including human, boar, bull, goat and rabbit species have provided further support for basic and clinical diagnosis.
Disclosure of Invention
The invention aims to obtain a simple and accurate method for identifying black-leaf monkey pregnancy. The applicant screens out a specific lectin through a large amount of analysis and experiments on urine glycoprotein sugar chains of black-leaf monkeys, and the specific lectin can be used for quickly identifying the specific glycoprotein sugar chains in urine so as to effectively identify the pregnancy conditions of the black-leaf monkeys.
The specific lectin probe combination is PHA-E, STL, PTL-I, PNA and SNA; the identification basis is as follows: when the sample detection result is that PHA-E, STL expression is up-regulated, and PTL-I, PNA and SNA expression are down-regulated, it indicates that the corresponding black-leaf monkeys are in gestation period at the time of sampling.
Here, the specific form of the "authentication basis" given by the relevant test product is not limited, for example: the above conditions are described in the attached product specifications; if software is involved, the basis can also be embodied by a corresponding algorithm.
The lectin probe combination can be used for preparing a kit for identifying black-leaf monkey pregnancy based on urine protein sugar chains, and the lectin probe combination is dotted on a lectin chip in the kit.
Furthermore, lectins RCA120 and MPL may be added to the lectin chip as internal references.
The method for identifying the pregnancy of the black-leaf monkey based on the urine protein glycoform by using the lectin probe combination comprises the following steps:
1) taking a cynomolgus monkey urine sample to be detected, and carrying out protein enrichment and fluorescence labeling;
2) taking the lectin chip, and sealing the lectin chip by using a sealing buffer solution; the lectin chip is prepared from at least five kinds of lectins selected from PHA-E, STL, PTL-I, PNA and SNA;
3) uniformly mixing the fluorescence-labeled urine protein with an incubation buffer solution, and then finishing incubation with the lectin chip;
4) and scanning the lectin chip, and analyzing whether the sugar chain expression corresponding to the three lectins is significantly changed or not, wherein if the sample detection result is that the expression of PHA-E, STL is up-regulated and the expression of PTL-I, PNA and SNA is down-regulated, the corresponding black-leaf monkey is in the gestation period when being sampled.
The invention has the advantages that:
the method is quick, simple, convenient and accurate for identifying the pregnancy condition of the black-leaf monkeys, and has lower implementation cost.
Drawings
FIG. 1 is a diagram showing the layout of lectin probes on an initially designed lectin chip;
FIG. 2 shows the fluorescence detection results of urine protein lectin chip for adult (P) and non-adult (NP) females; wherein (a) corresponds to a pregnant black-skinned monkey, (b) corresponds to a non-pregnant black-skinned monkey;
FIG. 3 shows two samples (a pregnant female F-3, a non-pregnant female F-7 for example) selected from FIG. 2, the positions of the 5 lectins on the chip, and the brightness comparison.
Detailed Description
The experiments and analyses related to the present application are described in detail below, and the specific development process of the inventors is not limited thereto.
1. Experimental part
1.1 reagents and materials
Glycine, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween-20 and protease inhibitors were purchased from Sigma-Aldrich, USA. Bovine Serum Albumin (BSA) was purchased from Merck, germany. Cy3 fluorescence was obtained from America Amersham. Other chemical reagents were of analytical grade and were not further purified prior to use. All experimental waters were ultrapure water treated with a Milli-Q50 pure water system (Millipore, USA). Sephadex G-25 column desalting was purchased from GE Healthcare, USA. Chip hybridization cassettes were purchased from Bio-Rad Bole, USA. Other common glassware is made in China. 37 kinds of agglutinin
1.2 Experimental instruments
Electrothermal blowing dry box, Tianjin Tester Co; autoclave, TOMY, Japan; an ultracentrifuge 5804R, Eppendorf, Germany; a micro nucleic acid protein analyzer, Implen, Germany; biochip scanner 4000B, Axon, USA; chip sample applicator, Bo ao crystal chip SmartArrayer48 sample applicator; chip hybridization chamber HL-2000, UVP, USA.
1.3 study subject and urine Collection
The black-leaf monkey urine sample is obtained from the protection research center of black-leaf monkeys in Sterculia, Guangxi province. Adult female black-leaf monkeys amounted to 15, with 5 pregnant females and 10 non-pregnant females, in the same location, and fed similarly sexually mature black-leaf monkeys.
Table 2: black leaf monkey urine sample information
| Numbering | Female (F)/male (M) | Pregnant (P)/Not Pregnant (NP) | Sample size/mL | Protein concentration mg/mL |
| F-1 | F | P | 50 | 4.83 |
| F-2 | F | P | 32 | 10.18 |
| F-3 | F | P | 30 | 6.41 |
| F-4 | F | P | 43 | 26.61 |
| F-5 | F | P | 59 | 9.00 |
| F-6 | F | NP | 37 | 17.59 |
| F-7 | F | NP | 28 | 12.17 |
| F-8 | F | NP | 30 | 7.46 |
| F-9 | F | NP | 30 | 12.21 |
| F-10 | F | NP | 35 | 3.89 |
| F-11 | F | NP | 26 | 5.99 |
| F-12 | F | NP | 58 | 4.92 |
| F-13 | F | NP | 31 | 9.66 |
| F-14 | F | NP | 30 | 6.92 |
| F-15 | F | NP | 25 | 8.92 |
1.4 urine mixing and fluorescence labeling
In order to eliminate the difference in collection time, samples collected from the same individual at different times were mixed, filtered through a 3KD ultrafiltration membrane to enrich the protein, and then quantified by Brandford. The mixed sample was labeled with Cy3 fluorochrome and free fluorescence was removed by Sephadex G-25 desalting column. The labeled protein is ready for lectin chip incubation.
1.5 lectin chips and data analysis
1.51 preparation of lectin chips
The slides were washed three times with absolute ethanol for 10min each time. And (5) spin-drying the glass slide in a centrifugal machine. Soaking in 250mL of 10% NaOH solution, keeping out of the sun, reacting on a shaking table for 12h, and then carrying out ultrasonic treatment for 15 min. Then, the mixture is washed with ultrapure water for four times, each time for 2min, and washed with absolute ethyl alcohol for two times, each time for 2 min. And (5) spin-drying. And then soaking the slide into 200mL of 10% GPTS solution, keeping out of the sun, incubating on a shaking table for 3h, and performing epoxidation modification on the slide. Ultrasonic cleaning for 15min, and cleaning with anhydrous ethanol for three times, each time for 10 min. Spin-drying, and placing the glass slide in a vacuum drying oven at 37 ℃ for drying for 3 h. And finally, placing the epoxy slide in a room-temperature dryer for storage for later use.
Designing a lectin chip matrix (figure 1), selecting 1mg/mL BSA as negative quality control, using Cy3 fluorescent dye-labeled BSA as position label, forming a12 x 10 matrix together with 37 lectins, wherein each lectin is spotted repeatedly 3 times, and each chip is repeated with 4 matrixes. The prepared sample solution is sequentially added into a 384-hole plate according to the matrix design sequence, and a pin point system of a Boo crystal core 48 sample applicator is used for preparing a chip on an epoxy modified film base. Incubating for 6h at room temperature and humidity of 55-65%, and vacuum drying the chip at 37 deg.C to fix the agglutinated cable on the chip. And (3) placing the prepared lectin chip in a drier at 4 ℃, and storing in a dark place for later use.
1.52 incubation of lectin chips and data analysis
(1) Encapsulation of lectin chips
Taking out the prepared lectin chip from a dryer at 4 ℃, returning to the temperature, firstly washing the glass slide once by PBST and PBS respectively, 5min each time, and centrifugally drying. The lectin chip was incubated with 700. mu.l of blocking buffer in a chip hybridization cassette and spun at 25 ℃ for 1 h. After the sealing is finished, the slide is washed twice by PBST and PBS respectively, each time is 5min, and the slide is dried. The chips after blocking were scanned with a Genepix4000B chip scanner and checked for blocking effect.
(2) Lectin chip detection of urine samples
And uniformly mixing 6 mu g of fluorescence labeled urine protein with an incubation buffer solution (the ratio is about 1:9), uniformly mixing each sample, uniformly adding 120 mu l of the mixture into a cover glass, covering a closed lectin chip, and rotationally incubating for 3 hours in a chip hybridization instrument at 25 ℃ in a dark place. After incubation, the slides were washed twice with PBST and PBS, each time for 5min, and centrifuged.
(3) Scanning and analysis of data
The chip was scanned using a Genepix4000B chip scanner, and the results are shown in FIG. 2. After the circle point analysis of the chip scanning result, the GenePix7.0 software derives a GPR file, and the analysis is carried out according to the data information in the GPR file. Values less than one time the background standard deviation in the raw data were subtracted, and the mean (AS) was then taken of the effective values of the three replicate points per sample per chip, with each set of mean values being the mean (AG) Standard Deviation (SDG) of the mean (AS) of each sample in the set.
2. Results section
The lectin chip is used for detecting urine of pregnant adult females and non-pregnant adult females respectively, chip data are obtained through professional software and normalized, and then two groups of results are compared, namely the Normalized Fluorescence Intensity (NFI) corresponding to each lectin obtains a Fold-change value in a pregnant group compared with a non-pregnant group. We believe that Fold-change >1.5 and Fold-change <0.67 are glycoprotein sugar chains that are up-and down-regulated in urine in adult pregnant females compared to non-pregnant females. And performing non-parametric Kruskal-Wallis test on lectin adjusted up and down in pregnancy group and non-pregnancy group with SPSS 20 to select significant lectin adjusted up and down (P <0.05)
(1)37 different lectins have different degrees of recognition on each group of urine glycoprotein sugar chains, which indicates that the glycoprotein sugar chains are expressed differently in the urine of adult pregnant females and non-adult pregnant females.
(2) Relative to non-pregnant black-leaf monkeys, the sugar chains recognized by 2 lectins PHA-E and STL appeared to be up-regulated in pregnant black-leaf monkey urine and were both significantly up-regulated; the 4 agglutinated PTL-I, PNA, SNA and LTL showed down-regulation, wherein PTL-I, PNA, SNA showed significant down-regulation.
TABLE 3 expression of urine glycoprotein profiles of pregnant and non-pregnant adult female black-leaf monkeys
The data in the table show that the lectin chip results correspond to the Fold-change values of pregnant NFI versus non-pregnant NFI. P is the adult pregnant female of all black-leaf monkeys, NP is the adult non-pregnant female of all black-leaf monkeys.
Based on the analysis results, the applicant also carries out multiple times of individual acquisition and detection in the later period, and the results are consistent with the results in the table 3 through observation and verification in a certain time.
Through screening of data in the table, a lectin with the Fold change of NFI larger than 1.5, smaller than 0.67 and P <0.05 is selected as a probe, RCA120 and MPL are used as internal references (in comparison of samples of any two groups, the ratio value is between 1.00 and 1.11, and a sugar chain recognized by the lectin is considered to be not different in the samples of the two groups), and a set of lectin probe combinations for identifying the pregnancy of the black-leaf monkeys are designed.
Table 4: lectin probe combination for identifying pregnancy
| Lectin | P/NP |
| PHA-E | ↑ |
| STL | ↑ |
| PTL-I | ↓ |
| PNA | ↓ |
| SNA | ↓ |
| RCA120 | - |
| MPL | - |
P is pregnant black-leaf monkey; NP is non-pregnant black-leaf monkey; ×) c: up-regulation of expression; ↓: down-regulation of expression; -: there was no significant difference. NFI fluorescence reference
When the lectin probe combination provided by the invention is used for actually detecting an individual, reference threshold values of the expression levels of the five lectins can be given in advance, or a black-leaf monkey sample which is known not to be pregnant is selected as a control sample to judge the expression up-regulation and down-regulation conditions.
And (3) recommending to collect a sample of the non-pregnant black-leaf monkey under the same environment as a control sample for each determination, removing environmental factors as far as possible, repeating the determined sample three times, and taking the average value to judge the expression up-regulation and down-regulation conditions.
The reference fluorescence signal values for the samples of non-pregnant black-leaf monkeys are as follows:
pregnancies up-regulated PHA-E: 0.025362, respectively; STL: 0.045465
Pregnancy Down-Regulation PTL-I: 0.008139, respectively; PNA: 0.012927, respectively; SNA: 0.012345.
Claims (5)
1. the application of the lectin probe combination in the aspect of identifying the pregnancy of black-leaved monkeys based on the urine protein glycoform is characterized in that: the lectin probe combination is a combination of PHA-E, STL, PTL-I, PNA and SNA; the identification basis is as follows: if the sample detection result is that the expression of PHA-E, STL is up-regulated, and the expression of PTL-I, PNA and SNA is down-regulated, it indicates that the corresponding black-leaf monkeys are in the gestation period at the time of sampling.
2. The lectin probe combination as claimed in claim 1, for use in identifying black-skinned monkey pregnancy based on the uroglycoform, characterized in that: the lectin probe combination also increases the expression downregulation of lectin LTL corresponding to gestation.
3. Use of the lectin probe combination as claimed in claim 1 for the preparation of a kit for identifying the pregnancy of black-skinned monkeys on the basis of the urine protein glycoform, wherein the lectin probe combination is spotted on a lectin chip in the kit.
4. Use according to claim 3, characterized in that: the lectin chip is spotted with RCA120 and/or MPL as internal reference.
5. A method for identifying black-leaved monkey pregnancy based on a urine protein glycoform is characterized by comprising the following steps:
1) taking a cynomolgus monkey urine sample to be detected, and carrying out protein enrichment and fluorescence labeling;
2) taking the lectin chip, and sealing the lectin chip by using a sealing buffer solution; the lectin chip is prepared from at least five kinds of lectins selected from PHA-E, STL, PTL-I, PNA and SNA;
3) uniformly mixing the fluorescence-labeled urine protein with an incubation buffer solution, and then finishing incubation with the lectin chip;
4) scanning the lectin chip, and analyzing whether the expression of sugar chains corresponding to the three lectins is significantly changed: if the sample detection result is that the expression of PHA-E, STL is up-regulated, and the expression of PTL-I, PNA and SNA is down-regulated, it indicates that the corresponding black-leaf monkeys are in the gestation period at the time of sampling.
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| CN105849278A (en) * | 2014-01-08 | 2016-08-10 | 李铮 | Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof |
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| CN105849278A (en) * | 2014-01-08 | 2016-08-10 | 李铮 | Lectin chip for identifying liver series of disease based on salivary glycoprotein sugar chain and application thereof |
| WO2017011876A1 (en) * | 2015-07-21 | 2017-01-26 | Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research | Glycoform biomarkers |
| CN109239363A (en) * | 2018-10-22 | 2019-01-18 | 西北大学 | A kind of application of the agglutinin probe combination in terms of identifying Activity budget gender based on Urine proteins sugar-type |
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