A kind of smcy sex-specific fusion antigen and its antibody and application

A recombinant antigen and polyclonal antibody technology, applied in the field of bioengineering, can solve the problem of time-consuming and labor-intensive methods for detecting gender by nucleic acid, and achieve the effect of significant technological progress.

Inactive Publication Date: 2019-07-26
SHANGHAI CRIMINAL SCI TECH RES INST +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a kind of SMCY sex-specific fusion antigen and its antibody and application, and the described a kind of SMCY sex-specific fusion antigen and its antibody and application will solve the problems in the prior art. Time-consuming and labor-intensive technical problems of using nucleic acid to detect gender

Method used

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  • A kind of smcy sex-specific fusion antigen and its antibody and application
  • A kind of smcy sex-specific fusion antigen and its antibody and application
  • A kind of smcy sex-specific fusion antigen and its antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 SMCY Sequence Alignment and Analysis

[0034] like figure 1 As shown, the amino acid sequences of the three variants of SMCY and SMCX were queried and downloaded from NCBI, and SCMY (1539aa) was compared with SMCX variant 1 (1560aa), variant2 (1379aa), and variant 3 (1559aa) using sequence analysis software. Sequence alignment revealed three different fragments:

[0035] Fragment 1: 256-293aa (DKTVHKKVTCPPTVTVKDEQSGGGNVSSTLLKQHLSL);

[0036] Fragment 2: 814-845aa (RLKNCLSEVEACIAQVLGLVSGQVARMDTPQL);

[0037] Fragment 3: 1467-1560aa (QKVDQGRNVENLVQQELQSKRARSSGIMSQVGREEEHYQEKADRENMFLTPSTDHSPFLKGNQNSLQHKDSGSSAACPSMPLLQLSYSDEQQL).

[0038] Using BIOSUN software to analyze the B cell epitope of the differential segment fusion antigen ( figure 2 ), the results showed that the fusion antigen still maintained the original B cell epitope and had good antigenicity.

Embodiment 2

[0039] The synthesis of embodiment 2 SMCY gene differential segment

[0040] According to the amino acid sequence of the three different fragments, the nucleic acid sequence is inversely deduced, codon optimization is performed, primers are designed according to the nucleic acid sequence, and then the synthetic primers are connected step by step through multiple rounds of PCR using the molecular biology bridging method to form A new SMCY gene recombinant encoding only these three fragments:

[0041] Composed of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, there is no connecting fragment between the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, so There is a connecting fragment between the amino acid sequences shown in EQ ID NO.2 and SEQ ID NO.3, and the connecting fragment is composed of two or three serines.

[0042] The homology to SMCX is significantly reduced, thereby greatly improving the male specificity of the SMCY sex-specif...

Embodiment 3

[0064] Example 3 Construction and expression of SMCY fusion antigen vector

[0065] Select the fusion segment antigen gene with a completely correct sequence and repeat it once, then digest it with BamH1 and EcoR1, recover the excised target fragment (about 516bp), and then connect it with the pET-28a vector that has been digested with BamH1 and EcoR1 with T4 Enzyme ligation to construct the recombinant expression plasmid pET-28a / SMCY ( image 3 , Figure 4 ). Then transform into Escherichia coli competent cells BL21(DE3), pick positive clone colonies, and inoculate LB culture medium containing kanamycin, after growing to the logarithmic phase, add IPTG, induce overnight at 37°C, and collect the bacterial liquid , SDS-PAGE electrophoresis to identify protein expression. The results showed that the SMCY sex-specific fusion antigen was expressed in the form of inclusion body, with a relative molecular weight of about 43KD ( Figure 5 ).

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Abstract

The present invention provides a recombinant antigen containing amino acid sequences shown as SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3. The invention also provides a colloidal gold rapid detection test paper strip, which comprises the recombinant antigen and a polyclonal antibody produced from the recombinant antigen. Fragments which are greatly different from SMCX (selected mouse cDNA on X) are screened aiming at the amino acid sequences of human SMCY (selected mouse cDNA on Y) antigen, the fragments are connected together by use of a molecular biology method for formation of a new SMCY gene recombinant, the homology of the SMCY gene recombinant and the SMCX is significantly reduced, and the male specificity of the SMCY (selected mouse cDNA on Y) sex specific fusion antigen is improved. A double-antibody sandwich ELISA detection technology is established based on acquisition of the specific antibody, and an on-site rapid detection method for sex determination by detection of the SMCY antigen of a human biological sample is established.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an antigen, in particular to an SMCY sex-specific fusion antigen, antibody and application thereof. Background technique [0002] Criminal cases urgently require us to provide technical support for the rapid detection of criminal cases by quickly identifying the characteristics of on-site physical evidence. At present, gender identification mainly uses the method of nucleic acid detection, but this detection method is time-consuming and laborious, and even misses the opportunity to solve the case. Therefore, a rapid on-site gender identification method is needed. [0003] The H-Y antigen is encoded by the Y chromosome and is a histocompatibility antigen unique to male animals. It was first discovered by Eichwald and Silmser in the allogeneic skin transplantation experiment with pure-line mice. H-Y antigens belong to a class of minor histocompatibility complexes, which are different f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K16/18C12N15/62G01N33/68G01N33/558
CPCC07K14/47C07K16/18C07K2319/00G01N33/558G01N33/68
Inventor 安志远周怀谷冯晓燕张贺秋
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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